Protease-activated t cell bispecific molecules

ABSTRACT

The present invention generally relates to novel protease-activatable T cell activating bispecific molecules and idiotype-specific polypeptides. The present invention also relates to polynucleotides encoding such protease-activatable T cell activating bispecific molecules and idiotype-specific polypeptides, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the protease-activatable T cell activating bispecific molecules and idiotype-specific polypeptides of the invention, and to methods of using these protease-activatable T cell activating bispecific molecules and idiotype-specific polypeptides in the treatment of disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.17/541,021, filed on Dec. 2, 2021, which is a divisional of U.S. patentapplication Ser. No. 16/138,417, filed on Sep. 21, 2018, now U.S. Pat.No. 11,242,390, which is a continuation of International PatentApplication No. PCT/EP2017/056556, filed on Mar. 20, 2017, which claimspriority to European Patent Application No. 16161740.2, filed on Mar.22, 2016, and to U.S. Patent Application No. 62/433,327, filed on Dec.13, 2016, the disclosures of which are incorporated herein by referencein their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 12, 2022, isnamed 51177-025003_Sequence_Listing_12_12_22_ST25 and is 309,204 bytesin size.

FIELD OF THE INVENTION

The present invention generally relates to novel protease-activatableantigen-binding molecules that comprise an anti-idiotype-binding moietywhich reversibly masks antigen binding of the molecule. Specifically,the invention relates to T cell binding molecules having ananti-idiotype-binding moiety that masks the CD3-binding moiety untilcleaved by a protease. This allows the CD3-binding moiety to beinaccessible or “masked” until it is in proximity to a target tissue,such as a tumor, e.g., tumor-infiltrating T cells. In addition, thepresent invention relates to polynucleotides encoding suchprotease-activated T cell binding molecules and idiotype-specificpolypeptides, and vectors and host cells comprising suchpolynucleotides. The invention further relates to methods for producingthe protease-activated T cell binding molecules of the invention, and tomethods of using the same, e.g., in the treatment of disease.

BACKGROUND

The selective destruction of an individual target cell or a specifictarget cell type is often desirable in a variety of clinical settings.For example, it is a primary goal of cancer therapy to specificallydestroy tumor cells, while leaving healthy cells and tissues intact andundamaged.

An attractive way of achieving this is by inducing an immune responseagainst the tumor, to make immune effector cells such as natural killer(NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumorcells. In this regard, bispecific antibodies designed to bind with one“arm” to a surface antigen on target cells, and with the second “arm” toan activating, invariant component of the T cell receptor (TCR) complex,have become of interest in recent years. The simultaneous binding ofsuch an antibody to both of its targets will force a temporaryinteraction between target cell and T cell, causing activation of anycytotoxic T cell and subsequent lysis of the target cell. Hence, theimmune response is re-directed to the target cells and is independent ofpeptide antigen presentation by the target cell or the specificity ofthe T cell as would be relevant for normal MHC-restricted activation ofCTLs.

In this context it is crucial that CTLs are activated only when in closeproximity to a target cell, i.e., the immunological synapse is mimicked.Particularly desirable are T cell activating bispecific molecules thatdo not require lymphocyte preconditioning or co-stimulation in order toelicit efficient lysis of target cells. Several bispecific antibodyformats have been developed and their suitability for T cell mediatedimmunotherapy investigated. These include BiTE (bispecific T cellengager) molecules (Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260(2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) andderivatives thereof, such as tandem diabodies (Kipriyanov et al., J MolBiol 293, 41-66 (1999)), DART (dual affinity retargeting) molecules,(Moore et al., Blood 117, 4542-51 (2011)), and triomabs (Seimetz et al.,Cancer Treat Rev 36, 458-467 (2010)).

The task of generating bispecific molecules suitable for treatmentprovides several technical challenges related to efficacy, toxicity,applicability and produceability that have to be met. In instances wherethe bispecific molecule targets an antigen on a target cell, e.g., acancer cell, that is also expressed in non-target tissue, toxicity canoccur. There is thus a need for efficacious T cell activating bispecificmolecules that unleash full T cell activation in the presence of targetcells but not in the presence of normal cells or tissue.

SUMMARY OF THE INVENTION

The invention generally relates to T cell activating bispecificmolecules that are activated selectively in the presence of a targetcell.

In one aspect, the invention relates to a protease-activatable T cellactivating bispecific molecule comprising

-   -   (a) a first antigen binding moiety capable of specific binding        to CD3;    -   (b) a second antigen binding moiety capable of specific binding        to a target cell antigen; and    -   (c) a masking moiety covalently attached to the T cell        bispecific binding molecule through a protease-cleavable linker,        wherein the masking moiety is capable of specific binding to the        idiotype of the first or the second antigen binding moiety        thereby reversibly concealing the first or second antigen        binding moiety.

In one embodiment, the masking moiety of the protease-activatable T cellactivating bispecific molecule is covalently attached to the firstantigen binding moiety. In one embodiment the masking moiety iscovalently attached to the heavy chain variable region of the firstantigen binding moiety. In one embodiment the masking moiety iscovalently attached to the light chain variable region of the firstantigen binding moiety. In one embodiment the masking moiety is ananti-idiotype scFv.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a second masking moiety reversibly concealing thesecond antigen binding moiety.

In one embodiment the protease capable of cleaving theprotease-cleavable linker is expressed by the target cell. In oneembodiment the second antigen binding moiety is a crossover Fab moleculewherein either the variable or the constant regions of the Fab lightchain and the Fab heavy chain are exchanged. In one embodiment thesecond antigen binding moiety is a crossover Fab molecule wherein theconstant regions of the Fab light chain and the Fab heavy chain areexchanged. In one embodiment the first antigen binding moiety is aconventional Fab molecule. In one embodiment the protease-activatable Tcell activating bispecific molecule comprises not more than one antigenbinding moiety capable of specific binding to CD3. In one embodiment theprotease-activatable T cell activating bispecific molecule comprises athird antigen binding moiety which is a Fab molecule capable of specificbinding to a target cell antigen. In one particular embodiment the thirdantigen binding moiety is identical to the second antigen bindingmoiety. In one particular embodiment the third antigen binding moiety isnot identical to the second antigen binding moiety. In one embodimentthe second antigen binding moiety is capable of specific binding toFolR1 or HER1. In one embodiment the second antigen binding moiety iscapable of specific binding to FolR1, HER1 or Mesothelin. In oneembodiment the second antigen binding moiety is capable of specificbinding to FolR1, HER1, HER2 or Mesothelin.

In one embodiment the first and the second antigen binding moiety arefused to each other, optionally via a peptide linker. In one particularembodiment the second antigen binding moiety is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding moiety. In one particular embodiment the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingmoiety. In one particular embodiment the Fab light chain of the firstantigen binding moiety and the Fab light chain of the second antigenbinding moiety are fused to each other, optionally via a peptide linker.

In one embodiment the protease-activatable T cell activating bispecificmolecule additionally comprises an Fc domain composed of a first and asecond subunit capable of stable association. In one embodiment the Fcdomain is an IgG, specifically an IgG₁ or IgG₄, Fc domain. In oneembodiment the Fc domain is a human Fc domain. In one embodiment the Fcdomain exhibits reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a native IgG₁ Fc domain. Inone embodiment the Fc domain comprises one or more amino acidsubstitution that reduces binding to an Fc receptor and/or effectorfunction. In one particular embodiment the one or more amino acidsubstitution is at one or more position selected from the group of L234,L235, and P329 (Kabat numbering). In one particular embodiment eachsubunit of the Fc domain comprises three amino acid substitutions thatreduce binding to an activating Fc receptor and/or effector functionwherein said amino acid substitutions are L234A, L235A and P329G. In oneparticular embodiment the Fc receptor is an Fey receptor. In oneembodiment the effector function is antibody-dependent cell-mediatedcytotoxicity (ADCC). In one embodiment, the target cell is a human cell.

In one embodiment the masking moiety comprises a heavy chain variableregion comprising at least one of:

-   -   (a) a heavy chain complementarity determining region (CDR H) 1        amino acid sequence of DYSIH (SEQ ID NO:20);    -   (b) a CDR H2 amino acid sequence of WINTETGEPAYADDFKG (SEQ ID        NO:21); and    -   (c) a CDR H3 amino acid sequence of PYDYDVLDY (SEQ ID NO:22).

In one embodiment the masking moiety comprises a light chain variableregion comprising at least one of:

-   -   (a) a light chain (CDR L)1 amino acid sequence of        RASKSVSTSNYSYIH (SEQ ID NO:23);    -   (b) a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24); and    -   (c) a CDR L3 amino acid sequence of QHSREFPWT (SEQ ID NO:25).

In one embodiment the masking moiety comprises a heavy chain variableregion comprising:

-   -   (a) a heavy chain complementarity determining region (CDR H) 1        amino acid sequence of DYSIH (SEQ ID NO:20);    -   (b) a CDR H2 amino acid sequence of WINTETGEPAYADDFKG (SEQ ID        NO:21);    -   (c) a CDR H3 amino acid sequence of PYDYDVLDY (SEQ ID NO:22);        and a light chain variable region comprising:    -   (d) a light chain (CDR L)1 amino acid sequence of        RASKSVSTSNYSYIH (SEQ ID NO:23);    -   (e) a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24); and    -   (f) a CDR L3 amino acid sequence of QHSREFPWT (SEQ ID NO:25).

In one embodiment the masking moiety comprises a heavy chain variableregion comprising at least one of:

-   -   (a) a heavy chain complementarity determining region (CDR H) 1        amino acid sequence of SYGVS (SEQ ID NO:26);    -   (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID        NO:27); and    -   (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID        NO:28).

In one embodiment the masking moiety comprises a light chain variableregion comprising at least one of:

-   -   (a) a light chain (CDR L)1 amino acid sequence of RASENIDSYLA        (SEQ ID NO:29);    -   (b) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30); and    -   (c) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO:31).

In one embodiment the masking moiety comprises a heavy chain variableregion comprising:

-   -   (a) a heavy chain complementarity determining region (CDR H) 1        amino acid sequence of SYGVS (SEQ ID NO:26);    -   (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID        NO:27);    -   (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID        NO:28); and a light chain variable region comprising:    -   (d) a light chain (CDR L)1 amino acid sequence of RASENIDSYLA        (SEQ ID NO:29);    -   (e) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30); and    -   (f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO:31).

In one embodiment the protease cleavable linker comprises at least oneprotease recognition sequence. In one embodiment the protease cleavablelinker comprises a protease recognition sequence. In one embodiment theprotease recognition sequence is selected from the group consisting of:

-   -   (a) RQARVVNG (SEQ ID NO:36);    -   (b) VHMPLGFLGPGRSRGSFP (SEQ ID NO:37);    -   (c) RQARVVNGXXXXXVPLSLYSG (SEQ ID NO:38); and    -   (d) RQARVVNGVPLSLYSG (SEQ ID NO:39)    -   (e) PLGLWSQ (SEQ ID NO:40), wherein X is any amino acid.

In one embodiment the protease cleavable linker comprises a proteaserecognition sequence. In one embodiment the protease recognitionsequence is selected from the group consisting of:

-   -   (a) RQARVVNG (SEQ ID NO:36);    -   (b) VHMPLGFLGPGRSRGSFP (SEQ ID NO:37);    -   (c) RQARVVNGXXXXXVPLSLYSG (SEQ ID NO:38);    -   (d) RQARVVNGVPLSLYSG (SEQ ID NO:39);    -   (e) PLGLWSQ (SEQ ID NO:40);    -   (f) VHMPLGFLGPRQARVVNG (SEQ ID NO:97);    -   (g) FVGGTG (SEQ ID NO:98);    -   (h) KKAAPVNG (SEQ ID NO:99);    -   (i) PMAKKVNG (SEQ ID NO:100);    -   (j) QARAKVNG (SEQ ID NO:101);    -   (k) VHMPLGFLGP (SEQ ID NO:102);    -   (l) QARAK (SEQ ID NO:103);    -   (m) VHMPLGFLGPPMAKK (SEQ ID NO:104);    -   (n) KKAAP (SEQ ID NO:105); and    -   (o) PMAKK (SEQ ID NO:106), wherein X is any amino acid.

In one embodiment the protease capable of cleaving theprotease-cleavable linker is selected from the group consisting ofmetalloproteinase, e.g., matrix metalloproteinase (MMP) 1-28 and ADisintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30 and33, serine protease, e.g., urokinase-type plasminogen activator andMatriptase, cysteine protease, aspartic protease, and cathepsinprotease. In one specific embodiment the protease is MMP9 or MMP2. In afurther specific embodiment, the protease is Matriptase. In oneembodiment the protease cleavable linker comprises the proteaserecognition sequence RQARVVNG (SEQ ID NO:36).

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the first antigen binding moietycomprises at least one heavy chain complementarity determining region(CDR) comprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46and at least one light chain CDR selected from the group of SEQ ID NO:17, SEQ ID NO: 18 and SEQ ID NO: 19.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the first antigen binding moietycomprises the heavy chain complementarity determining region (CDRs) ofSEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and the light chain CDRsof SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the first antigen binding moietycomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 43 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 55.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the first antigen binding moietycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 43 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 55.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietyis capable of specific binding to FolR1 and comprises at least one heavychain complementarity determining region (CDR) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one lightchain CDR comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO:19.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietyis capable of specific binding to FolR1 and comprises at least one heavychain complementarity determining region (CDR) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153 and at least onelight chain CDR comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 154, SEQ ID NO: 155 andSEQ ID NO: 156.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietyis capable of specific binding to FolR1 and comprises at least one heavychain complementarity determining region (CDR) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO:15 and SEQ ID NO: 16 and at least one light chain CDR selected from thegroup of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietyis capable of specific binding to FolR1 and comprises at least one heavychain complementarity determining region (CDR) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 151, SEQ IDNO: 152 and SEQ ID NO: 153 and at least one light chain CDR selectedfrom the group of SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietycomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 47 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 55.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietycomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 157 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 158.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 47 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 55.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 157 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 158.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietyis capable of specific binding to Mesothelin and comprises at least oneheavy chain complementarity determining region (CDR) comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109 and at least onelight chain CDR comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 110, SEQ ID NO: 111 andSEQ ID NO: 112.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietyis capable of specific binding to Mesothelin and comprises at least oneheavy chain complementarity determining region (CDR) comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 107, SEQID NO: 108 and SEQ ID NO: 109 and at least one light chain CDR selectedfrom the group of SEQ ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietycomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 113 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 114.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 113 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 114.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietyis capable of specific binding to HER1 and comprises at least one heavychain complementarity determining region (CDR) of any one of theantibodies disclosed in WO/2006/082515 incorporated herein by referencein its entirety.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietyis capable of specific binding to HER1 and comprises at least one heavychain complementarity determining region (CDR) comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and at least one lightchain CDR comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO:61.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietyis capable of specific binding to HER1 and comprises at least one heavychain complementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and atleast one light chain CDR selected from the group of SEQ ID NO: 59, SEQID NO: 60 and SEQ ID NO: 61.

In one embodiment of the protease-activatable T cell activatingbispecific molecule described herein the second antigen binding moietycomprises a heavy chain comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 32 and a light chain comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 33. In one embodimentof the protease-activatable T cell activating bispecific moleculedescribed herein the second antigen binding moiety comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 32 and a lightchain comprising the amino acid sequence of SEQ ID NO: 33.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, and the second and third antigen bindingmoieties are capable of specific binding to HER2, wherein the secondantigen binding moiety comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 160and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 161, wherein the third antigen bindingmoiety comprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 159 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 161.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) a first heavy chain comprising the amino acid sequence of        SEQ ID NO:2;    -   (b) a second heavy chain comprising the amino acid sequence of        SEQ ID NO:3; and    -   (c) a light chain comprising the amino acid sequence of SEQ ID        NO:1.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) a first heavy chain comprising the amino acid sequence of        SEQ ID NO:4;    -   (b) a second heavy chain comprising the amino acid sequence of        SEQ ID NO:3; and    -   (c) a light chain comprising the amino acid sequence of SEQ ID        NO:1.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) at least one heavy chain comprising the amino acid sequence        of SEQ ID NO:32;    -   (b) at least one light chain comprising the amino acid sequence        of SEQ ID NO:34.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) a first heavy chain comprising the amino acid sequence of        SEQ ID NO:72;    -   (b) a second heavy chain comprising the amino acid sequence of        SEQ ID NO:3; and    -   (c) a light chain comprising an amino acid sequence of SEQ ID        NO:1.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) a first heavy chain comprising the amino acid sequence of        SEQ ID NO:85;    -   (b) a second heavy chain comprising the amino acid sequence of        SEQ ID NO:3; and    -   (c) a light chain comprising an amino acid sequence of SEQ ID        NO:1.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) a first heavy chain comprising the amino acid sequence of        SEQ ID NO:73;    -   (b) a second heavy chain comprising the amino acid sequence of        SEQ ID NO:3;    -   (c) a first light chain comprising an amino acid sequence of SEQ        ID NO:1; and    -   (d) a second light chain comprising an amino acid sequence of        SEQ ID NO: 74.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) a first heavy chain comprising the amino acid sequence of        SEQ ID NO:77;    -   (b) a second heavy chain comprising the amino acid sequence of        SEQ ID NO:82;    -   (c) a first light chain comprising an amino acid sequence of SEQ        ID NO:78; and    -   (d) a second light chain comprising an amino acid sequence of        SEQ ID NO:81.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) a first heavy chain comprising the amino acid sequence of        SEQ ID NO:76;    -   (b) a second heavy chain comprising the amino acid sequence of        SEQ ID NO:77;    -   (c) a first light chain comprising an amino acid sequence of SEQ        ID NO:78; and    -   (d) a second light chain comprising an amino acid sequence of        SEQ ID NO:79.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) a first heavy chain comprising the amino acid sequence of        SEQ ID NO:132;    -   (b) a second heavy chain comprising the amino acid sequence of        SEQ ID NO:136;    -   (c) a first light chain comprising an amino acid sequence of SEQ        ID NO:81; and    -   (d) a second light chain comprising an amino acid sequence of        SEQ ID NO:133.

In one particular embodiment the protease-activatable T cell activatingbispecific molecule described herein comprises

-   -   (a) a first heavy chain comprising the amino acid sequence of        SEQ ID NO:137;    -   (b) a second heavy chain comprising the amino acid sequence of        SEQ ID NO:139;    -   (c) a first light chain comprising an amino acid sequence of SEQ        ID NO:81; and    -   (d) a second light chain comprising an amino acid sequence of        SEQ ID NO:138.

In one aspect, the invention relates to an idiotype-specific polypeptidefor reversibly concealing an anti-CD3 antigen binding site of amolecule. In one embodiment the idiotype-specific polypeptide is ananti-idiotype scFv. In one embodiment the idiotype-specific polypeptideis covalently attached to the molecule through a linker. In oneembodiment the linker is a peptide linker. In one embodiment the linkeris a protease-cleavable linker. In one embodiment the peptide linkercomprises at least one protease recognition site. In one embodiment theprotease is selected from the group consisting of metalloproteinase,e.g., matrix metalloproteinase (MMP) 1-28 and A Disintegrin AndMetalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serineprotease, e.g., urokinase-type plasminogen activator and Matriptase,cysteine protease, aspartic protease, and cathepsin protease. In onespecific embodiment the protease is MMP9 or MMP2. In a further specificembodiment, the protease is Matriptase. In one embodiment theidiotype-specific polypeptide is covalently attached to the moleculethrough more than one linker. In one embodiment the idiotype-specificpolypeptide is covalently attached to the molecule through two linkers.

In one embodiment the molecule which comprises the anti-CD3 antigenbinding site is a T-cell activating bispecific molecule. In oneparticular embodiment the idiotype-specific polypeptide comprises aheavy chain variable region comprising at least one of:

-   -   (a) a heavy chain complementarity determining region (CDR H) 1        amino acid sequence of DYSIH (SEQ ID NO:20);    -   (b) a CDR H2 amino acid sequence of WINTETGEPAYADDFKG (SEQ ID        NO:21); and    -   (c) a CDR H3 amino acid sequence of PYDYDVLDY (SEQ ID NO:22).

In one particular embodiment the idiotype-specific polypeptide comprisesa light chain variable region comprising at least one of:

-   -   (a) a light chain (CDR L)1 amino acid sequence of        RASKSVSTSNYSYIH (SEQ ID NO:23);    -   (b) a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24); and    -   (c) a CDR L3 amino acid sequence of QHSREFPWT (SEQ ID NO:25).

In one particular embodiment the idiotype-specific polypeptidecomprises:

-   -   (a) a heavy chain complementarity determining region (CDR H) 1        amino acid sequence of DYSIH (SEQ ID NO:20);    -   (b) a CDR H2 amino acid sequence of WINTETGEPAYADDFKG (SEQ ID        NO:21);    -   (c) a CDR H3 amino acid sequence of PYDYDVLDY (SEQ ID NO:22);        and a light chain variable region comprising:    -   (d) a light chain (CDR L)1 amino acid sequence of        RASKSVSTSNYSYIH (SEQ ID NO:23);    -   (e) a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24); and    -   (f) a CDR L3 amino acid sequence of QHSREFPWT (SEQ ID NO:25).

In one particular embodiment the idiotype-specific polypeptide comprisesa heavy chain variable region comprising at least one of:

-   -   (a) a heavy chain complementarity determining region (CDR H) 1        amino acid sequence of SYGVS (SEQ ID NO:26);    -   (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID        NO:27); and    -   (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID        NO:28).

In one particular embodiment the idiotype-specific polypeptide comprisesa light chain variable region comprising at least one of:

-   -   (a) a light chain (CDR L)1 amino acid sequence of RASENIDSYLA        (SEQ ID NO:29);    -   (b) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30); and    -   (c) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO:31).

In one particular embodiment the idiotype-specific polypeptide comprisesa heavy chain variable region comprising:

-   -   (a) a heavy chain complementarity determining region (CDR H) 1        amino acid sequence of SYGVS (SEQ ID NO:26);    -   (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID        NO:27);    -   (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID        NO:28); and a light chain variable region comprising:    -   (d) a light chain (CDR L)1 amino acid sequence of RASENIDSYLA        (SEQ ID NO:29);    -   (e) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30); and    -   (f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO:31).

According to another aspect of the invention, an isolated polynucleotideencoding a protease-activatable T cell activating bispecific molecule ofthe invention or a fragment thereof is provided. The invention alsoencompasses polypeptides encoded by the polynucleotides of theinvention. The invention further provides an expression vectorcomprising the isolated polynucleotide of the invention, and a host cellcomprising the isolated polynucleotide or the expression vector of theinvention. In some embodiments the host cell is a eukaryotic cell,particularly a mammalian cell. In another aspect is provided a method ofproducing the protease-activated T cell molecule of the invention,comprising the steps of a) culturing the host cell of the inventionunder conditions suitable for the expression of the protease-activated Tcell molecule and b) recovering the protease-activated T cell molecule.The invention also encompasses a protease-activated T cell moleculeproduced by the method of the invention.

In another aspect is provided a method of producing theidiotype-specific polypeptide of the invention, comprising the steps ofa) culturing the host cell of the invention under conditions suitablefor the expression of the protease-activated T cell molecule and b)recovering the idiotype-specific polypeptide. The invention alsoencompasses a idiotype-specific polypeptide produced by the method ofthe invention.

The invention further provides a pharmaceutical composition comprisingthe protease-activatable T cell activating bispecific molecule of theinvention and a pharmaceutically acceptable carrier.

Also encompassed by the invention are methods of using theprotease-activated T cell molecule and pharmaceutical composition of theinvention. In one aspect the invention provides a protease-activated Tcell molecule or a pharmaceutical composition of the invention for useas a medicament. In one aspect is provided a protease-activated T cellmolecule or a pharmaceutical composition according to the invention foruse in the treatment of a disease in an individual in need thereof. In aspecific embodiment the disease is cancer.

Also provided is the use of a protease-activated T cell molecule of theinvention for the manufacture of a medicament for the treatment of adisease in an individual in need thereof; as well as a method oftreating a disease in an individual, comprising administering to saidindividual a therapeutically effective amount of a compositioncomprising the protease-activated T cell molecule according to theinvention in a pharmaceutically acceptable form. In a specificembodiment the disease is cancer. In any of the above embodiments theindividual preferably is a mammal, particularly a human.

The invention also provides a method for inducing lysis of a targetcell, particularly a tumor cell, comprising contacting a target cellwith a protease-activated T cell molecule of the invention in thepresence of a T cell, particularly a cytotoxic T cell.

In another aspect the invention also provides a composition comprising aprotease-activatable T cell activating bispecific molecule describedherein and a pharmaceutically acceptable carrier. In another aspect theinvention also provides a composition comprising an idiotype-specificpolypeptide as described herein and a pharmaceutically acceptablecarrier.

In another aspect the invention also provides a protease-activatable Tcell activating bispecific molecule or an idiotype-specific polypeptideas described herein, or the composition described herein, for use as amedicament. In one embodiment the medicament is for treating or delayingprogression of cancer, treating or delaying progression of an immunerelated disease, and/or enhancing or stimulating an immune response orfunction in an individual.

In another aspect the invention also provides a protease-activatable Tcell activating bispecific molecule or idiotype-specific polypeptide asdescribed herein for use in the treatment of a disease in an individualin need thereof. In one embodiment, the disease is a proliferativedisorder, particularly cancer.

In another aspect the invention also provides a method of treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of a composition comprising theprotease-activatable T cell activating bispecific molecule orcomposition as described herein.

In another aspect the invention also provides a method for inducinglysis of a target cell, comprising contacting a target cell with theprotease-activatable T cell activating bispecific molecule orcomposition as described herein in the presence of a T cell. In oneembodiment the method for inducing lysis of a target cell is an in vitromethod. In one embodiment the target cell is a cancer cell. In oneembodiment the target cell expresses a protease capable of activatingthe protease-activatable T cell activating bispecific molecule.

In another aspect the invention also provides an anti-idiotype CD3antibody or antigen-binding fragment thereof specific for an idiotype ofan anti-CD3 antigen-binding molecule, wherein the anti-idiotype CD3antibody or fragment thereof when bound to the anti-CD3 antigen-bindingmolecule specifically blocks binding of the anti-CD3 antigen-bindingmolecule to CD3.

In one embodiment, the anti-idiotype CD3 antibody or antigen-bindingfragment thereof is reversibly associated with the anti-CD3antigen-binding molecule through a peptide linker comprising a proteaserecognition site. In one embodiment, the CD3 is a mouse, monkey or humanCD3.

In another aspect the invention provides a method of reducing in vivotoxicity of a T cell activating bispecific molecule comprising attachingan idiotype-specific polypeptide as described herein to the T cellactivating bispecific molecule with a protease-cleavable linker to forma protease-activatable T cell activating bispecific molecule, whereinthe protease-activatable T cell activating bispecific molecule hasreduced in vivo toxicity compared to the T cell activating bispecificmolecule.

SHORT DESCRIPTION OF THE FIGURES

FIGS. 1A-1E depict schematics of different CD3 binders with maskingmoieties. FIG. 1A: 7859 anti-ID CH2527 scFv 4.15.64 MK062 Matriptasesite CD3 Fc. FIG. 1B: 7860 anti-ID CH2527 scFv 4.32.63 MK062 Matriptasesite CD3 Fc. FIG. 1C: 7857 anti-ID CH2527 scFv 4.15.64 non-cleavablelinker CD3 Fc. FIG. 1D: ID 7858 anti-ID CH2527 scFv 4.32.63non-cleavable linker CD3 Fc. FIG. 1E: 7861 monovalent CD3 Fc.

FIG. 2 shows a table summarizing the affinities of the anti-idiotypicmasking moieties to the CD3 binder (CH2527).

FIGS. 3A-3D shows Capillary Electrophoresis-SDS analysis of themolecules depicted in FIGS. 1A and 1B. FIGS. 3A and 3B: CapillaryElectrophoresis-SDS analysis of the molecule depicted in FIG. 1A undernon reducing (FIG. 3A) and reducing conditions (FIG. 3B). Comparison ofthe untreated (I) and treated molecule (III) shows complete cleavage ofthe anti-ID scFv after rhMatriptase/ST14 treatment for 48 h at 37° C.One sample (II) was untreated but incubated at 37° C. for 48 h. FIGS. 3Cand 3D: Capillary Electrophoresis-SDS analysis of the molecule depictedin FIG. 1B under non-reducing (FIG. 3C) and reducing conditions (FIG.3D). Comparison of the untreated (I) and treated molecule (III) showscomplete cleavage of the anti-ID scFv after rhMatriptase/ST14 treatmentfor 48 h at 37° C. One sample (II) was untreated but incubated at 37° C.for 48 h.

FIGS. 4A-4C show the effect of anti-idiotypic masking of CD3 binding.FIGS. 4A and 4B depict results of Jurkat NFAT reporter assays to showthe masking effect of anti-idiotypic CD3 scFv 4.15.64 (FIG. 4A) oranti-idiotypic CD3 scFv 4.32.63 (FIG. 4B). Monovalent CD3 IgGs werecrosslinked via an anti-human Fc antibody (coated on assay plate) beforeJurkat NFAT (acute lymphatic leukemia reporter cell line with a NFATpromoter, expressing human CD3c) were added. The Jurkat-NFAT reportercell line (Promega) is a human acute lymphatic leukemia reporter cellline with a NFAT promoter, expressing human CD3c. If CD3 binder bindsCD3c Luciferase is expressed and this can be measured in Luminescenceafter addition of One-Glo substrate (Promega). FIG. 4C shows acomparison of EC50 values of CD3c binding for masked and unmaskedmonovalent CD3 binder.

FIGS. 5A-5H depict schematics of different T cell bispecific moleculeswith masking moieties. FIG. 5A: 7344 anti-ID CH2527 scFv 4.15.64 MK062Matriptase site CD3 16D5 Fc. FIG. 5B: 7676 anti-ID CH2527 scFv 4.15.64non-cleavable linker CD3 16D5 Fc. FIG. 5C: 7496 anti-ID CH2527 scFv4.32.63 MK062 Matriptase site CD3 16D5 Fc. FIG. 5D: 7611 anti-ID CH2527scFv 4.32.63 non-cleavable linker CD3 16D5 Fc. FIG. 5E: 6298GA916-D-16D5-02 sf W(1). FolR1 16D5 classic 2+1 TCB with common lightchain. FIG. 5F: 6100 GA916-D-16D5 sf W(3a). FolR1 16D5 inverted 2+1 TCBwith common light chain. FIG. 5G: ID 6182 DP47GS TCB sf CHO W(9a). DP47inverted 2+1 TCB. FIG. 5H: 7494 anti-ID CH2527 Fab 4.15.64 MK062Matriptase site CD3 16D5 Fc.

FIG. 6 shows a first plasmid ratios used for transfection by sizeexclusion chromatography (1(hole): 1 (knob): 3 (CLC)).

FIG. 7 shows a second plasmid ratios used for transfection by sizeexclusion chromatography. (1(hole): 2 (knob): 3 (CLC)).

FIG. 8 shows CE-SDS analysis of the TCB molecule depicted in FIG. 5A (ID7344) (final purified preparation): Lane A=non-reduced, lane B=reduced,lane C=Protein standard.

FIG. 9 shows CE-SDS analysis of the TCB molecule depicted in FIG. 5B (ID7676) (final purified preparation): Lane A=non-reduced, lane B=reduced,lane C=Protein standard.

FIG. 10 shows CE-SDS analysis of the TCB molecule depicted in FIG. 5C(ID 7496) (final purified preparation): Lane A=non-reduced, laneB=reduced, lane C=Protein standard.

FIG. 11 shows CE-SDS analysis of the TCB molecule depicted in FIG. 5D(ID 7611) (final purified preparation): Lane A=non-reduced, laneB=reduced, lane C=Protein standard.

FIG. 12A-12D show shows Capillary Electrophoresis-SDS analysis of themolecules depicted in FIGS. 5A and 5C. FIGS. 12A and 12B show CapillaryElectrophoresis of the molecules depicted in FIG. 5A (ID 7344) anti-IDCH2527 scFv 4.15.64 MK062 CD3 16D6 Fc under non reducing (FIG. 12A) andreducing conditions (FIG. 12B). Comparison of the untreated (I) andtreated molecule (III) shows complete cleavage of the anti-ID scFv afterrhMatriptase/ST14 treatment for 48 h at 37° C. One sample (II) wasuntreated but incubated at 37° C. for 48 h. Pre-stained protein Marker(IV) Mark 12 (Invitrogen) was used for estimation of correct moleculeweight. FIGS. 12C and 12D shows Capillary Electrophoresis of themolecule depicted in FIG. 5C (ID 7496) anti-ID CH2527 scFv 4.32.63 MK062CD3 16D6 Fc under non reducing (FIG. 12C) and reducing conditions (FIG.12D). Comparison of the untreated (I) and treated molecule (III) showscomplete cleavage of the anti-ID scFv after rhMatriptase/ST14 treatmentfor 48 h at 37° C. One sample (II) was untreated but incubated at 37° C.for 48 h. Pre-stained protein Marker (IV) Mark 12 (Invitrogen) was usedfor estimation of correct molecule weight.

FIG. 13 shows FolR1 expression level quantification done by Qifikit(Dako). Antibody for FolR1: #LS-C125620-100 (LifeSpan BioSciences Inc);used at 20 μg/ml; mouse IgG1 isotype: #554121 (BD).

FIGS. 14A and 14B show T cell activation by protease activated TCBs.FIG. 14A shows killing of Skov3 cells induced by protease-activated TCBmolecules at a concentration of 10 nM (TCBs with differentanti-idiotypic CD3 masks, cleavable and non-cleavable linker, moleculespre-treated with purified rhMatriptase/ST14) and human PBMCs after 48 hof incubation (E:T=7:1, effectors are human PBMCs). Pre-treatment withrhMatriptase/ST14 (R&D Systems) was done for 24 h at 37° C.

FIG. 14B shows T cell activation of human PBMCs induced by proteaseactivated TCB binding of 10 nM (TCBs with different anti-idiotypic CD3masks, cleavable and non-cleavable linker, treated molecules) on Skov3cells after 48 h of incubation (E:T=7:1, effectors are human PBMCs). Tcell activation markers CD25 (left panels) and CD69 (right panels). CD4+and CD8+ T cells as indicated.

FIGS. 15A and 15B show T cell activation by protease activated TCBs.FIG. 15A shows killing of Mkn-45 cells induced by protease activated TCBmolecules at a concentration of 100 nM (TCBs with differentanti-idiotypic CD3 masks, cleavable and non-cleavable linker, treatedmolecules) and human PBMCs after 48 h of incubation (E:T=7:1, effectorsare human PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) wasdone for 24 h at 37° C. FIG. 15B shows T cell activation of human PBMCsinduced by protease activated TCB binding of 100 nM (TCBs with differentanti-idiotypic CD3 masks, cleavable and non-cleavable linker, treatedmolecules) on Mkn-45 cells after 48 h of incubation (E:T=7:1, effectorsare human PBMCs). T cell activation markers CD25 (left panels) and CD69(right panels). CD4+ and CD8+ T cells as indicated.

FIG. 16 shows killing of HT29 cells induced by protease activated TCBmolecules at a concentration of 10 nM (TCBs with differentanti-idiotypic CD3 masks, cleavable and non-cleavable linker, treatedmolecules) and human PBMCs after 48 h of incubation (E:T=10:1, effectorsare human PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) wasdone for 24 h at 37° C. Bars from left to right are 7344: anti-ID CH2527scFv 4.15.64 MK062 CD3 16D6Fc; 7344: anti-ID CH2527 scFv 4.15.64 MK062CD3 16D6Fc_treated; 7676: anti-ID CH2527 scFv 4.15.64 non-cleavable CD316D6Fc; 7496 anti-ID CH2527 scFv 4.32.63 MK062 CD3 16D6 Fc; 7496 anti-IDCH2527 scFv 4.32.63 MK062 CD3 16D6 Fc_treated; 7611: ID anti CH2527 scFv4.32.63 non-cleavable linker CD3 16D6 Fc; 6298 GA916-D-16D5-02 sf W(1);6182 DP47GS TCB sf CHO W(9a).

FIG. 17 shows killing of Skov3 cells induced by protease activated TCBmolecules at a concentration of 10 nM (TCBs with differentanti-idiotypic CD3 masks, cleavable and non-cleavable linker, treatedmolecules) and human PBMCs (from a different donor than PBMCs used forFIG. 14A) after 48 h of incubation (E:T=10:1, effectors are humanPBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) was done for24 h at 37° C. Bars from left to right are 7344: anti-ID CH2527 scFv4.15.64 MK062 CD3 16D6Fc; 7344: anti-ID CH2527 scFv 4.15.64 MK062 CD316D6Fc_treated; 7676: anti-ID CH2527 scFv 4.15.64 non-cleavable CD316D6Fc; 7496 anti-ID CH2527 scFv 4.32.63 MK062 CD3 16D6 Fc; 7496 anti-IDCH2527 scFv 4.32.63 MK062 CD3 16D6 Fc_treated; 7611: ID anti CH2527 scFv4.32.63 non-cleavable linker CD3 16D6 Fc; 6298 GA916-D-16D5-02 sf W(1);6182 DP47GS TCB sf CHO W(9a).

FIGS. 18A and 18B show T cell activation by protease activated TCBs.FIG. 18A shows dose-dependent killing of HeLa cells induced by proteaseactivated TCB molecules (TCB with anti-idiotypic CD3 4.15.64 mask,cleavable and non-cleavable linker, treated molecule) and human PBMCs(isolated from buffy coat) after 48 h of incubation (E:T=10:1, effectorsare human PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) wasdone for 24 h at 37° C. FIG. 18B shows dose-dependent T cell activationof human PBMCs induced by protease activated TCB binding (TCB withanti-idiotypic CD3 4.15.64 mask, cleavable and non-cleavable linker,treated molecule) on HeLa cells after 48 h of incubation (E:T=10:1,effectors are human PBMCs). T cell activation markers CD25 (left panels)and CD69 (right panels). CD4+ and CD8+ T cells as indicated.

FIGS. 19A and 19B show T cell activation by protease activated TCBs.FIG. 19A shows dose-dependent killing of HeLa cells induced by proteaseactivated TCB molecules (TCB with anti-idiotypic CD3 4.32.63 mask,cleavable and non-cleavable linker, treated molecule) and human PBMCs(isolated from buffy coat) after 48 h of incubation (E:T=10:1, effectorsare human PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) wasdone for 24 h at 37° C. FIG. 19B shows dose-dependent T cell activationof human PBMCs induced by protease activated TCB binding (TCB withanti-idiotypic CD3 4.32.63 mask, cleavable and non-cleavable linker,treated molecule) on HeLa cells after 48 h of incubation (E:T=10:1,effectors are human PBMCs). T cell activation markers CD25 (left panels)and CD69 (right panels). CD4+ and CD8+ T cells as indicated.

FIGS. 20A and 20B show T cell activation by protease activated TCBs.FIG. 20A shows dose-dependent killing of Skov3 cells induced by proteaseactivated TCB molecules (TCB with anti-idiotypic CD3 4.15.64 mask,cleavable and non-cleavable linker, treated molecule) and human PBMCs(isolated from buffy coat) after 48 h of incubation (E:T=10:1, effectorsare human PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) wasdone for 24 h at 37° C. FIG. 20B shows dose-dependent T cell activationof human PBMCs induced by protease activated TCB binding (TCB withanti-idiotypic CD3 4.15.64 mask, cleavable and non-cleavable linker,treated molecule) on Skov3 cells after 48 h of incubation (E:T=10:1,effectors are human PBMCs). T cell activation markers CD25 (left panels)and CD69 (right panels). CD4+ and CD8+ T cells as indicated.

FIGS. 21A and 21B show T cell activation by protease activated TCBs.FIG. 21A shows dose-dependent killing of Skov3 cells induced by proteaseactivated TCB molecules (TCB with anti-idiotypic CD3 4.32.63 mask,cleavable and non-cleavable linker, treated molecule) and human PBMCs(isolated from buffy coat) after 48 h of incubation (E:T=10:1, effectorsare human PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) wasdone for 24 h at 37° C. FIG. 21B shows dose-dependent T cell activationof human PBMCs induced by protease activated TCB binding (TCB withanti-idiotypic CD3 4.32.63 mask, cleavable and non-cleavable linker,treated molecule) on Skov3 cells after 48 h of incubation (E:T=10:1,effectors are human PBMCs). T cell activation markers CD25 (left panels)and CD69 (right panels). CD4+ and CD8+ T cells as indicated.

FIGS. 22A and 22B show T cell activation by protease activated TCBs.FIG. 22A shows dose-dependent T cell activation of human PBMCs(different donor than in experiments described above) induced byprotease activated TCB binding (TCB with anti-idiotypic CD3 4.15.64mask, cleavable and non-cleavable linker, treated molecule) on HT29cells after 48 h of incubation (E:T=10:1, effectors are human PBMCs). Tcell activation markers CD25 (left panels) and CD69 (right panels). CD4+and CD8+ T cells as indicated. FIG. 22B shows dose-dependent T cellactivation of human PBMCs (different donor than in FIG. 16 ) induced byprotease activated TCB binding (TCB with anti-idiotypic CD3 4.32.63mask, cleavable and non-cleavable linker, treated molecule) on HT29cells after 48 h of incubation (E:T=10:1, effectors are human PBMCs). Tcell activation markers CD25 (left panels) and CD69 (right panels). CD4+and CD8+ T cells as indicated.

FIG. 23 shows dose-dependent T cell activation of human PBMCs (differentdonor than in experiments described above) induced by protease activatedTCB binding (TCB with anti-idiotypic CD3 4.15.64 mask, cleavable andnon-cleavable linker, treated molecule) on HRCEpiC cells after 48 h ofincubation (E:T=10:1, effectors are human PBMCs). T cell activationmarkers CD25 (left panels) and CD69 (right panels). CD4+ and CD8+ Tcells as indicated.

FIG. 24 shows killing of Ovcar3 cells induced by protease activated TCBmolecules at a concentration of 50 nM (TCBs with differentanti-idiotypic CD3 masks, cleavable and non-cleavable linker, treatedmolecules) and human PBMCs after 48 h of incubation (E:T=10:1, effectorsare human PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) wasdone for 10 min at 37° C. (not fully cleaved).

FIG. 25 shows killing of Skov3 cells induced by 10 nM of proteaseactivated TCB molecules (TCB with anti-idiotypic CD3 4.15.64 mask,cleavable and non-cleavable linker, treated molecule) and human PBMCs(isolated from buffy coat) after 48 h of incubation (E:T=10:1, effectorsare three different Donors for human PBMCs). Pre-treatment withrhMatriptase/ST14 (R&D Systems) was done for 24 h at 37° C.

FIG. 26 shows killing of Skov3 cells induced by 10 nM of proteaseactivated TCB molecules (TCB with anti-idiotypic CD3 4.32.63 mask,cleavable and non-cleavable linker, treated molecule) and human PBMCs(isolated from buffy coat) after 48 h of incubation (E:T=10:1, effectorsare three different Donors for human PBMCs). Pre-treatment withrhMatriptase/ST14 (R&D Systems) was done for 24 h at 37° C.

FIG. 27 shows killing of HeLa cells induced by 100 pM of proteaseactivated TCB molecules (TCB with anti-idiotypic CD3 4.32.63 mask,cleavable and non-cleavable linker, treated molecule) and human PBMCs(isolated from buffy coat) after 48 h of incubation (E:T=10:1, effectorsare three different Donors for human PBMCs). Pre-treatment withrhMatriptase/ST14 (R&D Systems) was done for 24 h at 37° C.

FIG. 28 depicts a schematic of anti-ID GA201 scFv Matrix Metalloproteasesite GA201 Fc (GA201-anti-GA201-scFv).

FIG. 29 depicts a schematics of the anti HER1 antibody GA201.

FIGS. 30A and 30B show capillary Electrophoresis-SDS analysis of themolecule depicted in FIG. 28 under non-reducing (FIG. 30A) and reducingconditions (FIG. 30B). The molecule depicted in FIG. 28 was purified tohomogeneity by Protein A and Size Exclusion chromatography and subjectedto Capillary electrophoresis-SDS analysis.

FIG. 31 shows FACS analysis of GA201-anti-GA201-scFv and GA201 bindingto HER1 expressed on H322M cells to confirm masking effect ofanti-idiotypic GA201 scFv. GA201-anti-GA201-scFv was incubated overnightwith the Matrix Metalloprotease MMP-2 and binding to H322M cells wascompared to uncleaved GA201-anti-GA201-scFv, GA201 and an isotype IgG1control antibody. Binding to HER1 on H322M cells was detected with aF(ab′)2-goat anti-human IgG Fc secondary antibody FITC conjugate andanalyzed by FACS using the BD FACS Canto II. The median fluorescenceintensity (MFI) was used for analysis.

FIG. 32 shows surface plasmon resonance analysis of HER1 binding ofmasked and unmasked GA201, before and after MMP2 cleavage.

FIGS. 33A-33J depict schematics of different T cell bispecific moleculeswith masking moieties.

FIG. 33A: ID 8364. 16D5 TCB, classic format, anti ID CH2527 scFv 4.32.63MMP9-MK062 Matriptase site N-terminally fused to CD3. FIG. 33B: ID 8363.16D5 TCB, classic format, anti ID CH2527 scFv 4.32.63 Cathepsin S/B siteN-terminally fused to CD3. FIG. 33C: ID 8365. 16D5 TCB, inverted format,anti ID CH2527 scFv 4.32.63 MK062 Matriptase site N-terminally fused tocommon light chain. FIG. 33D: ID 8366. 16D5 TCB, inverted format, antiID CH2527 scFv 4.32.63 non-cleavable linker N-terminally fused to commonlight chain. FIG. 33E: ID 8672. aMesothelin RG7787 charged residues TCB,classic format, anti ID CH2527 scFv 4.32.63 MMP9-MK062 Matriptase siteN-terminally fused to CD3×Fab. FIG. 33F: ID 8673. aMesothelin RG7787charged residues TCB, classic format, anti ID CH2527 scFv 4.32.63non-cleavable linker N-terminally fused to CD3× Fab. FIG. 33G: ID 8674.aMesothelin RG7787 charged residues TCB, inverted format, anti ID CH2527scFv 4.32.63 MMP9-MK062 Matriptase site N-terminally fused to CD3 XFab.FIG. 33H: 8675. aMesothelin RG7787 charged residues TCB, invertedformat, anti ID CH2527 scFv 4.32.63 non-cleavable linker N-terminallyfused to CD3 XFab. FIG. 33I: ID 8505. aMesothelin RG7787 chargedresidues CD3 XFab TCB, inverted format. FIG. 33J: ID 8676. CD3 XFabaMesothelin RG7787 charged residues TCB, classic format.

FIG. 34 depicts CE-SDS analysis of the TCB ID 8365 and TCB ID 8366(final purified preparation): Lane A=Protein standard, lane B=proteinstored at 4° C., lane C=protein pretreated with rhMatriptase/ST14 (R&DSystems), lane D=protein incubated for 72 h at 37° C. and laneE=molecule 3.

FIGS. 35A and 35B. depicts CE-SDS analysis of the TCB depicted in FIG.33A (ID 8364) and the TCB depicted in FIG. 33B (ID 8363). FIG. 35A:CE-SDS analysis of the TCB 8364 (final purified preparation): LaneA=Protein standard, lane B=protein stored at 4° C., lane C=proteinpretreated with rhMatriptase/ST14 (R&D Systems), lane D=proteinincubated for 72 h at 37° C. and lane E=non-cleavable linker construct.FIG. 35B: CE-SDS analysis of the TCB 8363 (final purified preparation):Lane A=Protein standard, lane B=protein stored at 4° C., lane C=proteinpretreated with rhCathepsin B (R&D Systems), lane D=protein pretreatedwith rhCathepsin S (R&D Systems), lane E=protein incubated for 72 h at37° C. and lane F=non-cleavable linker construct.

FIGS. 36A and 36B. depicts Jurkat NFAT activation assay using HeLa andSkov-3 cells as target cells. Each point represents the mean value oftriplicates. Standard deviation is indicated by error bars. Jurkat-NFATreporter cell line (Promega) is a human acute lymphatic leukemiareporter cell line with a NFAT promoter, expressing human CD3c. If theCD3 binder of the TCB binds the tumor target and the CD3 (cross-linkageis necessary) binds CD3c the Luciferase expression can be measured inLuminescence after addition of One-Glo substrate (Promega).

The FolR1 TCB (black triangles pointing down) and the pretreatedprotease activated TCB (8364, grey filled squares) with N-terminallyfused anti ID CD3 4.32.63 scFv and MMP9-Matriptase MK062 site werecompared. The molecule was treated with rhMatriptase/ST14 (R&D Systems)for about 20 h at 37° C. The masked TCB (containing a GS non-cleavablelinker, grey triangles pointing up) and the non-targeted TCB control(empty triangle pointing down) are shown as well. The dotted line showsthe Luminescence of target cells and effector cells without any TCB.

FIG. 36A shows a Jurkat NFAT activation assay using HeLa cells as targetcells.

FIG. 36B shows a Jurkat NFAT activation assay using Skov-3 cells astarget cells.

FIGS. 37A-37D depict tumor cell cytotoxicity mediated by FolR1 TCBs andhuman PBMCs (Effector:Target=10:1). Each point represents the mean valueof triplicates. Standard deviation is indicated by error bars. FIG. 37A:HeLa target cell cytotoxicity. Comparison of two different formats ofthe Protease activated TCBs both containing an anti idiotypic CD3 scFvlinked with a MK062 Matriptase linker. FIG. 37B: Skov-3 target cellcytotoxicity. Comparison of two different formats of the Proteaseactivated TCBs both containing an anti idiotypic CD3 scFv linked with aMK062 Matriptase linker. FIG. 37C: HeLa target cell cytotoxicity.Comparison of classic Protease activated TCB containing an antiidiotypic CD3 scFv and GS linkers with different protease sites.Protease activated TCB containing the MMP9-Matriptase MK062 linker(8364, grey squares), FolR1 TCB (light grey triangles pointing down),protease activated TCB containing only Matriptase MK062 (light greyrhomb)/Cathepsin site (grey circles) or non-cleavable linker (blacktriangles pointing down). FIG. 37D: Skov-3 target cell cytotoxicity.Comparison of classic Protease activated TCB containing an antiidiotypic CD3 scFv and GS linkers with different protease sites.Protease activated TCB containing the MMP9-Matriptase MK062 linker(8364, grey squares), FolR1 TCB (light grey triangles pointing down),protease activated TCB containing only Matriptase MK062 (light greyrhomb)/Cathepsin site (grey circles) or non-cleavable linker (blacktriangles pointing down).

FIGS. 38A and 38B depict quantification of CD69 of CD8 positive cellsafter co-incubation of primary human renal epithelial cortical cells(FIG. 38A) or human bronchial epithelial cells (FIG. 38B) with 200 nM ofthe different TCBs and three different donors of human PBMCs. T cellswere stained after 48 h of incubation. (E:T=10:1, effectors are humanPBMCs). Median fluorescence intensity of T cell activation marker CD69for CD8⁺ T cells is shown. Each point represents the mean value oftriplicates of three different human PBMC donors. Standard deviation isindicated in error bars. Unpaired t test was used for statisticalanalysis.

FIGS. 39A and 39B depict tumor cell cytotoxicity mediated by MSLN TCBsand human PBMCs (Effector:Target=10:1). Maximal lysis of the targetcells (=100%) was achieved by incubation of target cells with 1% TritonX-100 20 h before LDH readout. Minimal lysis (=0%) refers to targetcells co-incubated with effector cells without any TCB. Each pointrepresents the mean value of triplicates. Standard deviation isindicated by error bars. FIG. 39A: NCI H596 target cell cytotoxicity.Protease activated MSLN TCB containing an anti idiotypic CD3 scFv linkedwith a MMP9-MK062 Matriptase linker. The protease activated TCB (8672,light grey circles), the MSLN TCB (dark grey triangles pointing down)and the protease activated TCB containing a non-cleavable linker (8673,grey triangles pointing up) are compared. FIG. 39B: AsPC-1 target cellcytotoxicity. Protease activated MSLN TCB containing an anti idiotypicCD3 scFv linked with a MMP9-MK062 Matriptase linker. The proteaseactivated TCB (8672, light grey circles), the MSLN TCB (dark greytriangles pointing down) and the protease activated TCB containing anon-cleavable linker (8673, grey triangles pointing up) are compared.

FIG. 40 depicts a Jurkat-NFAT activation assay with primary tumorsamples and Protease activated FolR1 TCBs. Jurkat NFAT reporter cellsare activated after co-incubation with FolR1 TCB (6298) and Proteaseactivated FolR1 TCB containing MMP9-Matriptase cleavage site (8364).Protease activated FolR1 TCBs (8363, 8408) and control TCBs (8409, 7235)do not induce Luciferase expression. The dotted line indicates thebaseline Luminescence for Jurkat NFAT cells co-incubated with tumor.

FIGS. 41A-41C: Capillary electrophoresis of protease activated TCBsafter incubation in human serum. Molecules were incubated for 0 or 14days in human IgG depleted serum at 37° C. in a humidified incubator (5%CO₂). All molecules were purified by affinity chromatography (ProteinA)and then analyzed by Capillary electrophoresis. FIG. 41A: CE-SDSanalysis of serum, FolR1 TCB (6298) in serum at day 0 and day 14. FIG.41B: CE-SDS analysis of serum, Protease activated FolR1 TCB withMMP9-Matriptase linker (8364) in serum at day 0 and day 14. FIG. 41C:CE-SDS analysis of serum, Protease activated FolR1 TCB with Matriptaselinker (8408) in serum at day 0 and day 14 and the precleaved moleculein serum.

FIGS. 42A-42F depict schematics of different T cell bispecific moleculeswith masking moieties. FIG. 42A: ID 8955. Herceptarg TCB, classicformat, anti ID CH2527 scFv 4.32.63 MK062 MMP9 linker N-terminally fusedto VH. FIG. 42B: ID 8957. Herceptarg TCB, classic format, anti ID CH2527scFv 4.32.63 non cleavable linker N-terminally fused to VH. FIG. 42C: ID8959. Herceptarg TCB, classic format. FIG. 42D: ID 8997. FolR1 36F2 TCB,classic format, anti ID CH2527 scFv 4.32.63 MK062 MMP9 linkerN-terminally fused to VH. FIG. 42E: ID 8998. FolR1 36F2 TCB, classicformat, anti ID CH2527 scFv 4.32.63 non cleavable linker N-terminallyfused to VH. FIG. 42F: ID 8996. FolR1 36F2 TCB, classic format.

FIG. 43 depicts Human Bronchial Epithelial Cell toxicity mediated byhuman PBMCs and 100 nM or 10 nM of TCBs. Maximal lysis of the targetcells (=100%) was achieved by incubation of target cells with 1% TritonX-100 20 h before LDH readout. Minimal lysis (=0%) refers to targetcells co-incubated with effector cells without any TCB. Each pointrepresents the mean value of triplicates. Standard deviation isindicated by error bars.

FIG. 44 depicts FolR1 negative target cell (Mkn-45) cytotoxicitymediated by 100 nM of FolR1 TCBs and human PBMCs. Maximal lysis of thetarget cells (=100%) was achieved by incubation of target cells with 1%Triton X-100 20 h before LDH readout. Minimal lysis (=0%) refers totarget cells co-incubated with effector cells without any TCB. Eachpoint represents the mean value of triplicates. Standard deviation isindicated by error bars.

FIG. 45A to FIG. 45N depict schematic diagrams of exemplary antibodyconstructs.

DETAILED DESCRIPTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are immunoglobulinsand derivatives, e.g., fragments, thereof.

The term “bispecific” means that the antigen binding molecule is able tospecifically bind to at least two distinct antigenic determinants.Typically, a bispecific antigen binding molecule comprises two antigenbinding sites, each of which is specific for a different antigenicdeterminant. In certain embodiments the bispecific antigen bindingmolecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antigen binding molecule. As such,the term “monovalent binding to an antigen” denotes the presence of one(and not more than one) antigen binding site specific for the antigen inthe antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more aminoacid residues, of an antigen binding molecule which provides interactionwith the antigen. For example, the antigen binding site of an antibodycomprises amino acid residues from the complementarity determiningregions (CDRs). A native immunoglobulin molecule typically has twoantigen binding sites, a Fab molecule typically has a single antigenbinding site.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g., a second antigenbinding moiety) to a target site, for example to a specific type oftumor cell or tumor stroma bearing the antigenic determinant. In anotherembodiment an antigen binding moiety is able to activate signalingthrough its target antigen, for example a T cell receptor complexantigen. Antigen binding moieties include antibodies and fragmentsthereof as further defined herein. Particular antigen binding moietiesinclude an antigen binding domain of an antibody, comprising an antibodyheavy chain variable region and an antibody light chain variable region.In certain embodiments, the antigen binding moieties may compriseantibody constant regions as further defined herein and known in theart. Useful heavy chain constant regions include any of the fiveisotypes: α, δ, ε, γ, or μ. Useful light chain constant regions includeany of the two isotypes: κ and λ.

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g., a contiguousstretch of amino acids or a conformational configuration made up ofdifferent regions of non-contiguous amino acids) on a polypeptidemacromolecule to which an antigen binding moiety binds, forming anantigen binding moiety-antigen complex. Useful antigenic determinantscan be found, for example, on the surfaces of tumor cells, on thesurfaces of virus-infected cells, on the surfaces of other diseasedcells, on the surface of immune cells, free in blood serum, and/or inthe extracellular matrix (ECM). The proteins referred to as antigensherein (e.g., FolR1, HER1, HER2, CD3, Mesothelin) can be any native formof the proteins from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated. In a particular embodiment the antigen is a humanprotein. Where reference is made to a specific protein herein, the termencompasses the “full-length”, unprocessed protein as well as any formof the protein that results from processing in the cell. The term alsoencompasses naturally occurring variants of the protein, e.g., splicevariants or allelic variants. Exemplary human proteins useful asantigens include, but are not limited to: FolR1, HER1 and CD3,particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version130), NCBI RefSeq no. NP_000724.1, SEQ ID NO: 54 for the human sequence;or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1 for thecynomolgus [Macaca fascicularis] sequence). In certain embodiments theprotease-activatable T cell activating bispecific molecule of theinvention binds to an epitope of CD3 or a target cell antigen that isconserved among the CD3 or target antigen from different species. Incertain embodiments the protease-activatable T cell activatingbispecific molecule of the invention binds to CD3 and FolR1, but doesnot bind to FolR2 or FolR3. In certain embodiments theprotease-activatable T cell activating bispecific molecule of theinvention binds to CD3 and HER1. In certain embodiments theprotease-activatable T cell activating bispecific molecule of theinvention binds to CD3 and Mesothelin. In certain embodiments theprotease-activatable T cell activating bispecific molecule of theinvention binds to CD3 and HER2. By “specific binding” is meant that thebinding is selective for the antigen and can be discriminated fromunwanted or non-specific interactions. The ability of an antigen bindingmoiety to bind to a specific antigenic determinant can be measuredeither through an enzyme-linked immunosorbent assay (ELISA) or othertechniques familiar to one of skill in the art, e.g., surface plasmonresonance (SPR) technique (analyzed on a BIAcore instrument) (Liljebladet al., Glyco J 17, 323-329 (2000)), and traditional binding assays(Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extentof binding of an antigen binding moiety to an unrelated protein is lessthan about 10% of the binding of the antigen binding moiety to theantigen as measured, e.g., by SPR. In certain embodiments, an antigenbinding moiety that binds to the antigen, or an antigen binding moleculecomprising that antigen binding moiety, has a dissociation constant(K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001nM (e.g., 10⁻⁸M or less, e.g., from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to10⁻¹³ M).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., an antigen binding moiety and an antigen, or areceptor and its ligand). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (K_(D)), whichis the ratio of dissociation and association rate constants (k_(off) andk_(on), respectively). Thus, equivalent affinities may comprisedifferent rate constants, as long as the ratio of the rate constantsremains the same. Affinity can be measured by well-established methodsknown in the art, including those described herein. A particular methodfor measuring affinity is Surface Plasmon Resonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. The protease-activatable T cell activatingbispecific molecules of the invention are capable of inducing T cellactivation. Suitable assays to measure T cell activation are known inthe art described herein.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma.

As used herein, the terms “first” and “second” with respect to antigenbinding moieties etc., are used for convenience of distinguishing whenthere is more than one of each type of moiety. Use of these terms is notintended to confer a specific order or orientation of theprotease-activatable T cell activating bispecific molecule unlessexplicitly so stated.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By “fused” is meant that the components (e.g., a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds. In certainembodiments, one of the antigen binding moieties is a single-chain Fabmolecule, i.e. a Fab molecule wherein the Fab light chain and the Fabheavy chain are connected by a peptide linker to form a single peptidechain. In a particular such embodiment, the C-terminus of the Fab lightchain is connected to the N-terminus of the Fab heavy chain in thesingle-chain Fab molecule.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein either the variable regions or the constant regions ofthe Fab heavy and light chain are exchanged, i.e. the crossover Fabmolecule comprises a peptide chain composed of the light chain variableregion and the heavy chain constant region, and a peptide chain composedof the heavy chain variable region and the light chain constant region.For clarity, in a crossover Fab molecule wherein the variable regions ofthe Fab light chain and the Fab heavy chain are exchanged, the peptidechain comprising the heavy chain constant region is referred to hereinas the “heavy chain” of the crossover Fab molecule. Conversely, in acrossover Fab molecule wherein the constant regions of the Fab lightchain and the Fab heavy chain are exchanged, the peptide chaincomprising the heavy chain variable region is referred to herein as the“heavy chain” of the crossover Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant regions (VH-CH1), and a lightchain composed of the light chain variable and constant regions (VL-CL).

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable region (VH), also called a variable heavy domain or a heavychain variable domain, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g., γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, and antibody fragments so long asthey exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2, diabodies, linear antibodies, single-chain antibody molecules(e.g., scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g., Plückthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)2 fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g., U.S. Pat. No. 6,248,516 B 1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g., E. coli or phage), as described herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, Sequences of Proteins of ImmunologicalInterest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987),where the definitions include overlapping or subsets of amino acidresidues when compared against each other. Nevertheless, application ofeither definition to refer to a CDR of an antibody or variants thereofis intended to be within the scope of the term as defined and usedherein. The appropriate amino acid residues which encompass the CDRs asdefined by each of the above cited references are set forth below inTable 1 as a comparison. The exact residue numbers which encompass aparticular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues comprisea particular CDR given the variable region amino acid sequence of theantibody.

TABLE 1 CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table 1 refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system. The polypeptide sequences ofthe sequence listing are not numbered according to the Kabat numberingsystem. However, it is well within the ordinary skill of one in the artto convert the numbering of the sequences of the Sequence Listing toKabat numbering.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, theC-terminal lysine (Lys447) of the Fc region may or may not be present.Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991. A “subunit”of an Fc domain as used herein refers to one of the two polypeptidesforming the dimeric Fc domain, i.e. a polypeptide comprising C-terminalconstant regions of an immunoglobulin heavy chain, capable of stableself-association. For example, a subunit of an IgG Fc domain comprisesan IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g., antigen bindingmoieties) are not the same. In some embodiments the modificationpromoting association comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g., B cell receptor), and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Particularamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g., the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G₃₂₉, P329G, or Pro329Gly.

As used herein, term “polypeptide” refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain of two ormore amino acids, and does not refer to a specific length of theproduct. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain of two or more amino acids, are included within the definition of“polypeptide,” and the term “polypeptide” may be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide may be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Itmay be generated in any manner, including by chemical synthesis. Apolypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded.

By an “isolated” polypeptide or a variant, or derivative thereof isintended a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan be removed from its native or natural environment. Recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g., messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g., DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator. By a nucleic acid or polynucleotide having a nucleotidesequence at least, for example, 95% “identical” to a referencenucleotide sequence of the present invention, it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These alterations of the reference sequence mayoccur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence. As a practicalmatter, whether any particular polynucleotide sequence is at least 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequenceof the present invention can be determined conventionally using knowncomputer programs, such as the ones discussed above for polypeptides(e.g., ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Hostcells include cultured cells, e.g., mammalian cultured cells, such asCHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeastcells, insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc domain of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Humanactivating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa(CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orderivatives thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “reduced ADCC” is defined as either a reduction in thenumber of target cells that are lysed in a given time, at a givenconcentration of antibody in the medium surrounding the target cells, bythe mechanism of ADCC defined above, and/or an increase in theconcentration of antibody in the medium surrounding the target cells,required to achieve the lysis of a given number of target cells in agiven time, by the mechanism of ADCC. The reduction in ADCC is relativeto the ADCC mediated by the same antibody produced by the same type ofhost cells, using the same standard production, purification,formulation and storage methods (which are known to those skilled in theart), but that has not been engineered. For example the reduction inADCC mediated by an antibody comprising in its Fc domain an amino acidsubstitution that reduces ADCC, is relative to the ADCC mediated by thesame antibody without this amino acid substitution in the Fc domain.Suitable assays to measure ADCC are well known in the art (see e.g., PCTpublication no. WO 2006/082515 or PCT publication no. WO 2012/130831).

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g., a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments,protease-activatable T cell activating bispecific molecules of theinvention are used to delay development of a disease or to slow theprogression of a disease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

An “idiotype-specific polypeptide” as used herein refers to apolypeptide that recognizes the idiotype of an antigen-binding moiety,e.g., an antigen-binding moiety specific for CD3. The idiotype-specificpolypeptide is capable of specifically binding to the variable region ofthe antigen-binding moiety and thereby reducing or preventing specificbinding of the antigen-binding moiety to its cognate antigen. Whenassociated with a molecule that comprises the antigen-binding moiety,the idiotype-specific polypeptide can function as a masking moiety ofthe molecule. Specifically disclosed herein are anti-idiotype antibodiesor anti-idiotype-binding antibody fragments specific for the idiotype ofanti-CD3 binding molecules.

“Protease” or “proteolytic enzyme” as used herein refers to anyproteolytic enzyme that cleaves the linker at a recognition site andthat is expressed by a target cell. Such proteases might be secreted bythe target cell or remain associated with the target cell, e.g., on thetarget cell surface. Examples of proteases include but are not limitedto metalloproteinases, e.g., matrix metalloproteinase 1-28 and ADisintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30 and33, serine proteases, e.g., urokinase-type plasminogen activator andMatriptase, cysteine protease, aspartic proteases, and members of thecathepsin family.

“Protease activatable” as used herein, with respect to the T cellactivating bispecific molecule, refers to a T cell activating bispecificmolecule having reduced or abrogated ability to activate T cells due toa masking moiety that reduces or abrogates the T cell activatingbispecific molecule's ability to bind to CD3. Upon dissociation of themasking moiety by proteolytic cleavage, e.g., by proteolytic cleavage ofa linker connecting the masking moiety to the T cell activatingbispecific molecule, binding to CD3 is restored and the T cellactivating bispecific molecule is thereby activated.

“Reversibly concealing” as used herein refers to the binding of amasking moiety or idiotype-specific polypeptide to an antigen-bindingmoiety or molecule such as to prevent the antigen-binding moiety ormolecule from its antigen, e.g., CD3. This concealing is reversible inthat the idiotype-specific polypeptide can be released from theantigen-binding moiety or molecule, e.g., by protease cleavage, andthereby freeing the antigen-binding moiety or molecule to bind to itsantigen.

DETAILED DESCRIPTION

In one aspect, the invention relates to a protease-activatable T cellactivating bispecific molecule comprising

-   -   (a) a first antigen binding moiety capable of specific binding        to CD3;    -   (b) a second antigen binding moiety capable of specific binding        to a target cell antigen; and    -   (c) a masking moiety covalently attached to the T cell        bispecific binding molecule through a protease-cleavable linker,        wherein the masking moiety is capable of specific binding to the        idiotype of the first or the second antigen binding moiety        thereby reversibly concealing the first or second antigen        binding moiety.

The first antigen binding moiety capable of specific binding to CD3comprises an idiotype. In one embodiment, the masking moiety of theprotease-activatable T cell activating bispecific molecule is covalentlyattached to the first antigen binding moiety. In one embodiment themasking moiety is covalently attached to the heavy chain variable regionof the first antigen binding moiety. In one embodiment the maskingmoiety is covalently attached to the light chain variable region of thefirst antigen binding moiety. This covalent bond is separate from thespecific binding, which is preferably non-covalent, of the maskingmoiety to the idiotype first antigen binding site. The idiotype of thefirst antigen binding moiety comprises its variable region. In oneembodiment the masking moiety binds to amino acid residues that makecontact with CD3 when the first antigen biding moiety is bound to CD3.In a preferred embodiment, the masking moiety is not the cognate antigenor fragments thereof of the first antigen binding moiety, i.e., themasking moiety is not a CD3 or fragments thereof. In one embodiment themasking moiety is an anti-idiotypic antibody or fragment thereof. In oneembodiment, the masking moiety is an anti-idiotypic scFv. Exemplaryembodiments of masking moieties which are anti-idiotypic scFv, andprotease activatable T cell activating molecules comprising such maskingmoieties, are described in detail in the examples. In one embodiment theprotease-activatable T cell activating bispecific molecule comprises asecond masking moiety reversibly concealing the second antigen bindingmoiety.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, and which comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to a target cell antigen.

In one embodiment the first antigen binding moiety comprises a heavychain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acidsequence of SEQ ID NO: 43 and a light chain variable region comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to an amino acid sequence of SEQ ID NO: 55.

In one embodiment the first antigen binding moiety comprises the heavychain variable region comprising an amino acid sequence of SEQ ID NO: 43and the light chain variable region comprising an amino acid sequence ofSEQ ID NO: 55.

In a specific embodiment the second antigen binding moiety is capable ofspecific binding to FolR1 and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18 and SEQ ID NO: 19.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to FolR1 and comprises a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 47 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 55.

In a specific embodiment the second antigen binding moiety is capable ofspecific binding to FolR1 and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153 and atleast one light chain CDR selected from the group of SEQ ID NO: 154, SEQID NO: 155 and SEQ ID NO: 156.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to FolR1 and comprises a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 157 and a light chain variable region comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 158.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to HER1 and comprises at least one heavychain complementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and atleast one light chain CDR selected from the group of SEQ ID NO: 59, SEQID NO: 60 and SEQ ID NO: 61.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to HER1 and comprises a heavy chaincomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO: 32,and a light chain comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acidsequence of SEQ ID NO: 33.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding to HER1 and comprises a heavy chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 115 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 116.

In a specific embodiment the second antigen binding moiety is capable ofspecific binding to Mesothelin and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109 and atleast one light chain CDR selected from the group of SEQ ID NO: 110, SEQID NO: 111 and SEQ ID NO: 112.

In another specific embodiment, the second antigen binding moiety iscapable of specific binding Mesothelin and comprises a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 113 and a light chain variable region comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 114. In oneembodiment the present invention provides a protease-activatable T cellactivating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to FolR1 comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18 and SEQ ID NO: 19.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55,(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to FolR1 comprising heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to FolR1 comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153 and atleast one light chain CDR selected from the group of SEQ ID NO: 154, SEQID NO: 155 and SEQ ID NO: 156.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55,(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to FolR1 comprising heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 157and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 158.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to HER1 comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and atleast one light chain CDR selected from the group of SEQ ID NO: 59, SEQID NO: 60 and SEQ ID NO: 61.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55.(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to HER1 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 115and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 116.

In a particular embodiment, the first antigen binding moiety is acrossover Fab molecule wherein either the variable or the constantregions of the Fab light chain and the Fab heavy chain are exchanged.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19;(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Mesothelin comprising at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109 and atleast one light chain CDR selected from the group of SEQ ID NO: 110, SEQID NO: 111 and SEQ ID NO: 112.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55,(ii) a second antigen binding moiety which is a Fab molecule capable ofspecific binding to Mesothelin comprising heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 113and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 114.

In one embodiment, the second antigen binding moiety is a conventionalFab molecule.

In a particular embodiment, the first antigen binding moiety is acrossover Fab molecule wherein the constant regions of the Fab lightchain and the Fab heavy chain are exchanged, and the second antigenbinding moiety is a conventional Fab molecule. In a further particularembodiment, the first and the second antigen binding moiety are fused toeach other, optionally through a peptide linker.

In particular embodiments, the protease-activatable T cell activatingbispecific molecule further comprises an Fc domain composed of a firstand a second subunit capable of stable association. In a furtherparticular embodiment, not more than one antigen binding moiety capableof specific binding to CD3 is present in the protease-activatable T cellactivating bispecific molecule (i.e. the protease-activatable T cellactivating bispecific molecule provides monovalent binding to CD3).

Protease-Activatable T Cell Activating Bispecific Molecule Formats

The components of the protease-activatable T cell activating bispecificmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIGS. 1A-1E and 5A-5H. Furtherexemplary configurations are depicted in FIGS. 33A-33K.

In particular embodiments, the protease-activatable T cell activatingbispecific molecule comprises an Fc domain composed of a first and asecond subunit capable of stable association. In some embodiments, thesecond antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the first or the second subunit of theFc domain.

In one such embodiment, the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second antigen binding moiety. In a specific suchembodiment, the protease-activatable T cell activating bispecificmolecule essentially consists of a first and a second antigen bindingmoiety, an Fc domain composed of a first and a second subunit, andoptionally one or more peptide linkers, wherein the first antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding moiety,and the second antigen binding moiety is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Optionally, the Fab light chain of the first antigenbinding moiety and the Fab light chain of the second antigen bindingmoiety may additionally be fused to each other.

In another such embodiment, the first antigen binding moiety is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a specific such embodiment, theprotease-activatable T cell activating bispecific molecule essentiallyconsists of a first and a second antigen binding moiety, an Fc domaincomposed of a first and a second subunit, and optionally one or morepeptide linkers, wherein the first and the second antigen binding moietyare each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain.

In other embodiments, the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain.

In a particular such embodiment, the second antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding moiety. In a specific suchembodiment, the protease-activatable T cell activating bispecificmolecule essentially consists of a first and a second antigen bindingmoiety, an Fc domain composed of a first and a second subunit, andoptionally one or more peptide linkers, wherein the second antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding moiety,and the first antigen binding moiety is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Optionally, the Fab light chain of the first antigenbinding moiety and the Fab light chain of the second antigen bindingmoiety may additionally be fused to each other.

The antigen binding moieties may be fused to the Fc domain or to eachother directly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) orG₄(SG₄)_(n) peptide linkers. “n” is generally a number between 1 and 10,typically between 2 and 4. A particularly suitable peptide linker forfusing the Fab light chains of the first and the second antigen bindingmoiety to each other is (G₄S)₂. An exemplary peptide linker suitable forconnecting the Fab heavy chains of the first and the second antigenbinding moiety is EPKSC(D)-(G₄S)₂ (SEQ ID NOs 105 and 106).Additionally, linkers may comprise (a portion of) an immunoglobulinhinge region. Particularly where an antigen binding moiety is fused tothe N-terminus of an Fc domain subunit, it may be fused via animmunoglobulin hinge region or a portion thereof, with or without anadditional peptide linker.

A protease-activatable T cell activating bispecific molecule with asingle antigen binding moiety capable of specific binding to a targetcell antigen is useful, particularly in cases where internalization ofthe target cell antigen is to be expected following binding of a highaffinity antigen binding moiety. In such cases, the presence of morethan one antigen binding moiety specific for the target cell antigen mayenhance internalization of the target cell antigen, thereby reducing itsavailability.

In many other cases, however, it will be advantageous to have aprotease-activatable T cell activating bispecific molecule comprisingtwo or more antigen binding moieties specific for a target cell antigen(see examples in shown in FIGS. 5A-5H), for example to optimizetargeting to the target site or to allow crosslinking of target cellantigens.

Accordingly, in certain embodiments, the protease-activatable T cellactivating bispecific molecule of the invention further comprises athird antigen binding moiety which is a Fab molecule capable of specificbinding to a target cell antigen. In one embodiment, the third antigenbinding moiety is a conventional Fab molecule. In one embodiment, thethird antigen binding moiety is capable of specific binding to the sametarget cell antigen as the second antigen binding moiety. In aparticular embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and the second and third antigen bindingmoieties are capable of specific binding to a target cell antigen. In aparticular embodiment, the second and the third antigen binding moietyare identical (i.e. they comprise the same amino acid sequences).

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and the second and third antigen bindingmoieties are capable of specific binding to FolR1, wherein the secondand third antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18 and SEQ ID NO: 19.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19; and the second and third antigen bindingmoieties are capable of specific binding to FolR1, wherein the secondand third antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18 and SEQ ID NO: 19.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19; and the second and third antigen bindingmoieties are capable of specific binding to FolR1, wherein the secondand third antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18 and SEQ ID NO: 19.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, and the second and third antigen bindingmoieties are capable of specific binding to FolR1, wherein the secondand third antigen binding moieties comprise a heavy chain variableregion comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 47 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 55.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and the second and third antigen bindingmoieties are capable of specific binding to HER1, wherein the second andthird antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and atleast one light chain CDR selected from the group of SEQ ID NO: 59, SEQID NO: 60 and SEQ ID NO: 61.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19; and the second and third antigen bindingmoieties are capable of specific binding to HER1, wherein the second andthird antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and atleast one light chain CDR selected from the group of SEQ ID NO: 59, SEQID NO: 60 and SEQ ID NO: 61.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18 and SEQ ID NO: 19; and the second and third antigen bindingmoieties are capable of specific binding to HER1, wherein the second andthird antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and atleast one light chain CDR selected from the group of SEQ ID NO: 59, SEQID NO: 60 and SEQ ID NO: 61.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, and the second and third antigen bindingmoieties are capable of specific binding to HER1, wherein the second andthird antigen binding moieties comprise a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 115and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 116. In one embodiment, the first antigenbinding moiety is capable of specific binding to CD3, and the second andthird antigen binding moieties are capable of specific binding to HER2,wherein the second antigen binding moiety comprises at least one heavychain complementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144 and atleast one light chain CDR selected from the group of SEQ ID NO: 148, SEQID NO: 149 and SEQ ID NO: 150, and wherein the third antigen bindingmoiety comprises at least one heavy chain complementarity determiningregion (CDR) selected from the group consisting of SEQ ID NO: 145, SEQID NO: 146 and SEQ ID NO: 147 and at least one light chain CDR selectedfrom the group of SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19; and the second and third antigen bindingmoieties are capable of specific binding to HER2, wherein the secondantigen binding moiety comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID NO: 144 and atleast one light chain CDR selected from the group of SEQ ID NO: 148, SEQID NO: 149 and SEQ ID NO: 150, and wherein the third antigen bindingmoiety comprises at least one heavy chain complementarity determiningregion (CDR) selected from the group consisting of SEQ ID NO: 145, SEQID NO: 146 and SEQ ID NO: 147 and at least one light chain CDR selectedfrom the group of SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, and the second and third antigen bindingmoieties are capable of specific binding to HER2, wherein the secondantigen binding moiety comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 160and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 161, wherein the third antigen bindingmoiety comprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 159 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 161.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and the second and third antigen bindingmoieties are capable of specific binding to Mesothelin, wherein thesecond and third antigen binding moieties comprise at least one heavychain complementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109 and atleast one light chain CDR selected from the group of SEQ ID NO: 110, SEQID NO: 111 and SEQ ID NO: 112.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19; and the second and third antigen bindingmoieties are capable of specific binding to Mesothelin, wherein thesecond and third antigen binding moieties comprise at least one heavychain complementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109 and atleast one light chain CDR selected from the group of SEQ ID NO: 110, SEQID NO: 111 and SEQ ID NO: 112.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43,and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, and the second and third antigen bindingmoieties are capable of specific binding to Mesothelin, wherein thesecond and third antigen binding moieties comprise a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 113 and a light chain variable region comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 114.

In one embodiment, the first antigen binding moiety is capable ofspecific binding to CD3, and the second and third antigen bindingmoieties are capable of specific binding to HER1, wherein the second andthird antigen binding moieties comprise at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and atleast one light chain CDR selected from the group consisting of SEQ IDNO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.

In a particular embodiment, the first antigen binding moiety is capableof specific binding to CD3, and comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and atleast one light chain CDR selected from the group consisting of SEQ IDNO: 17, SEQ ID NO: 18 and SEQ ID NO: 19; and the second and thirdantigen binding moieties are capable of specific binding to HER1,wherein the second and third antigen binding moieties comprise at leastone heavy chain complementarity determining region (CDR) selected fromthe group consisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58and at least one light chain CDR selected from the group consisting ofSEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.

In one embodiment, the third antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a more specific embodiment, thesecond and the third antigen binding moiety are each fused at theC-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain, and the first antigen binding moiety is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the second antigen binding moiety. Optionally, the Fablight chain of the first antigen binding moiety and the Fab light chainof the second antigen binding moiety may additionally be fused to eachother.

The second and the third antigen binding moiety may be fused to the Fcdomain directly or through a peptide linker. In a particular embodimentthe second and the third antigen binding moiety are each fused to the Fcdomain through an immunoglobulin hinge region. In a specific embodiment,the immunoglobulin hinge region is a human IgG₁ hinge region. In oneembodiment the second and the third antigen binding moiety and the Fcdomain are part of an immunoglobulin molecule. In a particularembodiment the immunoglobulin molecule is an IgG class immunoglobulin.In an even more particular embodiment the immunoglobulin is an IgG₁subclass immunoglobulin. In another embodiment the immunoglobulin is anIgG₄ subclass immunoglobulin. In a further particular embodiment theimmunoglobulin is a human immunoglobulin. In other embodiments theimmunoglobulin is a chimeric immunoglobulin or a humanizedimmunoglobulin. In one embodiment, the protease-activatable T cellactivating bispecific molecule essentially consists of an immunoglobulinmolecule capable of specific binding to a target cell antigen, and anantigen binding moiety capable of specific binding to CD3 wherein theantigen binding moiety is a Fab molecule, particularly a crossover Fabmolecule, fused to the N-terminus of one of the immunoglobulin heavychains, optionally via a peptide linker.

In a particular embodiment, the first and the third antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the secondantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingmoiety. In a specific such embodiment, the protease-activatable T cellactivating bispecific molecule essentially consists of a first, a secondand a third antigen binding moiety, an Fc domain composed of a first anda second subunit, and optionally one or more peptide linkers, whereinthe second antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety, and the first antigen binding moiety is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and wherein the third antigen binding moietyis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe second subunit of the Fc domain. Optionally, the Fab light chain ofthe first antigen binding moiety and the Fab light chain of the secondantigen binding moiety may additionally be fused to each other.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising the heavy chain complementaritydetermining region (CDR) 1 of SEQ ID NO: 44, the heavy chain CDR 2 ofSEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID NO: 46, the light chainCDR 1 of SEQ ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and thelight chain CDR 3 of SEQ ID NO: 19, wherein the first antigen bindingmoiety is a crossover Fab molecule wherein either the variable or theconstant regions, particularly the constant regions, of the Fab lightchain and the Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to FolR1 comprising the heavy chainCDR 1 of SEQ ID NO: 14, the heavy chain CDR 2 of SEQ ID NO: 15, theheavy chain CDR 3 of SEQ ID NO: 16, the light chain CDR 1 of SEQ ID NO:17, the light chain CDR 2 of SEQ ID NO: 18 and the light chain CDR3 ofSEQ ID NO: 19.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, wherein the first antigen binding moietyis a crossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to FolR1 comprising heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 47 and a light chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 55.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising the heavy chain complementaritydetermining region (CDR) 1 of SEQ ID NO: 44, the heavy chain CDR 2 ofSEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID NO: 46, the light chainCDR 1 of SEQ ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and thelight chain CDR 3 of SEQ ID NO: 19, wherein the first antigen bindingmoiety is a crossover Fab molecule wherein either the variable or theconstant regions, particularly the constant regions, of the Fab lightchain and the Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to FolR1 comprising the heavy chainCDR 1 of SEQ ID NO: 151, the heavy chain CDR 2 of SEQ ID NO: 152, theheavy chain CDR 3 of SEQ ID NO: 153, the light chain CDR 1 of SEQ ID NO:154, the light chain CDR 2 of SEQ ID NO: 155 and the light chain CDR3 ofSEQ ID NO: 156.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, wherein the first antigen binding moietyis a crossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to FolR1 comprising heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 157 and a light chain variable region comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 158.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising the heavy chain complementaritydetermining region (CDR) 1 of SEQ ID NO: 44, the heavy chain CDR 2 ofSEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID NO: 46, the light chainCDR 1 of SEQ ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and thelight chain CDR 3 of SEQ ID NO: 19, wherein the first antigen bindingmoiety is a crossover Fab molecule wherein either the variable or theconstant regions, particularly the constant regions, of the Fab lightchain and the Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to HER1 comprising the heavy chainCDR 1 of SEQ ID NO: 56, the heavy chain CDR 2 of SEQ ID NO: 57, theheavy chain CDR 3 of SEQ ID NO: 58, the light chain CDR 1 of SEQ ID NO:59, the light chain CDR 2 of SEQ ID NO: 60 and the light chain CDR3 ofSEQ ID NO: 61.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, wherein the first antigen binding moietyis a crossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to HER1 comprising a heavy chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 115 and a light chain variable region comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 116.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising the heavy chain complementaritydetermining region (CDR) 1 of SEQ ID NO: 44, the heavy chain CDR 2 ofSEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID NO: 46, the light chainCDR 1 of SEQ ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and thelight chain CDR 3 of SEQ ID NO: 19, wherein the first antigen bindingmoiety is a crossover Fab molecule wherein either the variable or theconstant regions, particularly the constant regions, of the Fab lightchain and the Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to HER2, wherein the second antigenbinding moiety comprises the heavy chain CDR 1 of SEQ ID NO: 142, theheavy chain CDR 2 of SEQ ID NO: 143, the heavy chain CDR 3 of SEQ ID NO:144, the light chain CDR 1 of SEQ ID NO: 148, the light chain CDR 2 ofSEQ ID NO: 149 and the light chain CDR3 of SEQ ID NO: 150, and whereinthe third antigen binding moiety comprises the heavy chain CDR 1 of SEQID NO: 145, the heavy chain CDR 2 of SEQ ID NO: 146, the heavy chain CDR3 of SEQ ID NO: 148, the light chain CDR 1 of SEQ ID NO: 148, the lightchain CDR 2 of SEQ ID NO: 149 and the light chain CDR3 of SEQ ID NO:150.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, wherein the first antigen binding moietyis a crossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to HER2, wherein the second antigenbinding moiety comprises a heavy chain variable region comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 160 and a lightchain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 161, and wherein the third antigen binding moietycomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 159 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 161.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3, comprising the heavy chain complementaritydetermining region (CDR) 1 of SEQ ID NO: 44, the heavy chain CDR 2 ofSEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID NO: 46, the light chainCDR 1 of SEQ ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and thelight chain CDR 3 of SEQ ID NO: 19, wherein the first antigen bindingmoiety is a crossover Fab molecule wherein either the variable or theconstant regions, particularly the constant regions, of the Fab lightchain and the Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to Mesothelin comprising the heavychain CDR 1 of SEQ ID NO: 107, the heavy chain CDR 2 of SEQ ID NO: 108,the heavy chain CDR 3 of SEQ ID NO: 109, the light chain CDR 1 of SEQ IDNO: 110, the light chain CDR 2 of SEQ ID NO: 111 and the light chainCDR3 of SEQ ID NO: 112.

In one embodiment the present invention provides a protease-activatableT cell activating bispecific molecule comprising

(i) a first antigen binding moiety which is a Fab molecule capable ofspecific binding to CD3 comprising a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 43and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 55, wherein the first antigen binding moietyis a crossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding moiety each of which is a Fabmolecule capable of specific binding to Mesothelin comprising heavychain variable region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 113 and a light chain variable region comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 114.

The protease-activatable T cell activating bispecific molecule accordingto any of the ten above embodiments may further comprise (iii) an Fcdomain composed of a first and a second subunit capable of stableassociation, wherein the second antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding moiety, and the first antigen bindingmoiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the second subunit of the Fc domain.

In some of the protease-activatable T cell activating bispecificmolecule of the invention, the Fab light chain of the first antigenbinding moiety and the Fab light chain of the second antigen bindingmoiety are fused to each other, optionally via a linker peptide.Depending on the configuration of the first and the second antigenbinding moiety, the Fab light chain of the first antigen binding moietymay be fused at its C-terminus to the N-terminus of the Fab light chainof the second antigen binding moiety, or the Fab light chain of thesecond antigen binding moiety may be fused at its C-terminus to theN-terminus of the Fab light chain of the first antigen binding moiety.Fusion of the Fab light chains of the first and the second antigenbinding moiety further reduces mispairing of unmatched Fab heavy andlight chains, and also reduces the number of plasmids needed forexpression of some of the protease-activatable T cell activatingbispecific molecule of the invention. In certain embodiments theprotease-activatable T cell activating bispecific molecule comprises apolypeptide wherein the Fab light chain variable region of the firstantigen binding moiety shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the first antigen binding moiety(i.e. a the first antigen binding moiety comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VL₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)), and apolypeptide wherein a the Fab heavy chain of the second antigen bindingmoiety shares a carboxy-terminal peptide bond with an Fc domain subunit(VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). In some embodiments theprotease-activatable T cell activating bispecific molecule furthercomprises a polypeptide wherein the Fab heavy chain variable region ofthe first antigen binding moiety shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the first antigen bindingmoiety (VH₍₁₎-CL₍₁₎ and the Fab light chain polypeptide of the secondantigen binding moiety (VL₍₂₎-CL₍₂₎). In certain embodiments thepolypeptides are covalently linked, e.g., by a disulfide bond.

In alternative embodiments the protease-activatable T cell activatingbispecific molecule comprises a polypeptide wherein the Fab heavy chainvariable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first antigen binding moiety (i.e. the first antigen bindingmoiety comprises a crossover Fab heavy chain, wherein the heavy chainconstant region is replaced by a light chain constant region), which inturn shares a carboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CL₍₁₎-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavychain of the second antigen binding moiety shares a carboxy-terminalpeptide bond with an Fc domain subunit (VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). Insome embodiments the protease-activatable T cell activating bispecificmolecule further comprises a polypeptide wherein the Fab light chainvariable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the first antigen binding moiety (VL₍₁₎-CH1₍₀₎ and the Fab lightchain polypeptide of the second antigen binding moiety (VL₍₂₎-CL₍₂₎). Incertain embodiments the polypeptides are covalently linked, e.g., by adisulfide bond.

In some embodiments, the protease-activatable T cell activatingbispecific molecule comprises a polypeptide wherein the Fab light chainvariable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the first antigen binding moiety (i.e. the first antigen bindingmoiety comprises a crossover Fab heavy chain, wherein the heavy chainvariable region is replaced by a light chain variable region), which inturn shares a carboxy-terminal peptide bond with the Fab heavy chain ofthe second antigen binding moiety, which in turn shares acarboxy-terminal peptide bond with an Fc domain subunit(VL₍₁₎-CH1₍₁₎-VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). In other embodiments, theprotease-activatable T cell activating bispecific molecule comprises apolypeptide wherein the Fab heavy chain variable region of the firstantigen binding moiety shares a carboxy-terminal peptide bond with theFab light chain constant region of the first antigen binding moiety(i.e. the first antigen binding moiety comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the second antigen binding moiety,which in turn shares a carboxy-terminal peptide bond with an Fc domainsubunit (VH₍₁₎-CL₍₁₎-VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). In still otherembodiments, the protease-activatable T cell activating bispecificmolecule comprises a polypeptide wherein the Fab heavy chain of thesecond antigen binding moiety shares a carboxy-terminal peptide bondwith the Fab light chain variable region of the first antigen bindingmoiety which in turn shares a carboxy-terminal peptide bond with the Fabheavy chain constant region of the first antigen binding moiety (i.e.the first antigen binding moiety comprises a crossover Fab heavy chain,wherein the heavy chain variable region is replaced by a light chainvariable region), which in turn shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₂₎-CH1₍₂₎-VL₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). Inother embodiments, the protease-activatable T cell activating bispecificmolecule comprises a polypeptide wherein the Fab heavy chain of thesecond antigen binding moiety shares a carboxy-terminal peptide bondwith the Fab heavy chain variable region of the first antigen bindingmoiety which in turn shares a carboxy-terminal peptide bond with the Fablight chain constant region of the first antigen binding moiety (i.e.the first antigen binding moiety comprises a crossover Fab heavy chain,wherein the heavy chain constant region is replaced by a light chainconstant region), which in turn shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₂₎-CH1₍₂₎-VH₍₁₎-CL₍₁₎-CH2-CH3(-CH4)).

In some of these embodiments the protease-activatable T cell activatingbispecific molecule further comprises a crossover Fab light chainpolypeptide of the first antigen binding moiety, wherein the Fab heavychain variable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first antigen binding moiety (VH₍₁₎-CL₍₁₎), and the Fab lightchain polypeptide of the second antigen binding moiety (VL₍₂₎-CL₍₂₎). Inothers of these embodiments the protease-activatable T cell activatingbispecific molecule further comprises a crossover Fab light chainpolypeptide, wherein the Fab light chain variable region of the firstantigen binding moiety shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the first antigen binding moiety(VL₍₁₎-CH1₍₀₎, and the Fab light chain polypeptide of the second antigenbinding moiety (VL₍₂₎-CL₍₂₎). In still others of these embodiments theprotease-activatable T cell activating bispecific molecule furthercomprises a polypeptide wherein the Fab light chain variable region ofthe first antigen binding moiety shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the first antigen bindingmoiety which in turn shares a carboxy-terminal peptide bond with the Fablight chain polypeptide of the second antigen binding moiety(VL₍₁₎-CH1₍₁₎-VL₍₂₎-CL₍₂₎), a polypeptide wherein the Fab heavy chainvariable region of the first antigen binding moiety shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first antigen binding moiety which in turn shares acarboxy-terminal peptide bond with the Fab light chain polypeptide ofthe second antigen binding moiety (VH₍₁₎-CL₍₁₎-VL₍₂₎-CL₍₂₎), apolypeptide wherein the Fab light chain polypeptide of the secondantigen binding moiety shares a carboxy-terminal peptide bond with theFab light chain variable region of the first antigen binding moietywhich in turn shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the first antigen binding moiety(VL₍₂₎-CL₍₂₎-VL₍₁₎-CH1₍₀₎, or a polypeptide wherein the Fab light chainpolypeptide of the second antigen binding moiety shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the first antigen binding moiety which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first antigen binding moiety (VL₍₂₎-CL₍₂₎-VH₍₁₎-CL₍₁₎).

The protease-activatable T cell activating bispecific molecule accordingto these embodiments may further comprise (i) an Fc domain subunitpolypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavychain of a third antigen binding moiety shares a carboxy-terminalpeptide bond with an Fc domain subunit (VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) andthe Fab light chain polypeptide of a third antigen binding moiety(VL₍₃₎-CL₍₃₎). In certain embodiments the polypeptides are covalentlylinked, e.g., by a disulfide bond.

According to any of the above embodiments, components of theprotease-activatable T cell activating bispecific molecule (e.g.,antigen binding moiety, Fc domain) may be fused directly or throughvarious linkers, particularly peptide linkers comprising one or moreamino acids, typically about 2-20 amino acids, that are described hereinor are known in the art. Suitable, non-immunogenic peptide linkersinclude, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) or G₄(SG₄)_(n)peptide linkers, wherein n is generally a number between 1 and 10,typically between 2 and 4.

Fc Domain

The Fc domain of the protease-activatable T cell activating bispecificmolecule consists of a pair of polypeptide chains comprising heavy chaindomains of an immunoglobulin molecule. For example, the Fc domain of animmunoglobulin G (IgG) molecule is a dimer, each subunit of whichcomprises the CH2 and CH3 IgG heavy chain constant domains. The twosubunits of the Fc domain are capable of stable association with eachother. In one embodiment the protease-activatable T cell activatingbispecific molecule of the invention comprises not more than one Fcdomain.

In one embodiment according the invention the Fc domain of theprotease-activatable T cell activating bispecific molecule is an IgG Fcdomain. In a particular embodiment the Fc domain is an IgG₁ Fc domain.In another embodiment the Fc domain is an IgG4 Fc domain. In a morespecific embodiment, the Fc domain is an IgG4 Fc domain comprising anamino acid substitution at position S228 (Kabat numbering), particularlythe amino acid substitution S228P. This amino acid substitution reducesin vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al.,Drug Metabolism and Disposition 38, 84-91 (2010)). In a furtherparticular embodiment the Fc domain is human.

Fc Domain Modifications Promoting Heterodimerization

Protease-activatable T cell activating bispecific molecules according tothe invention comprise different antigen binding moieties, fused to oneor the other of the two subunits of the Fc domain, thus the two subunitsof the Fc domain are typically comprised in two non-identicalpolypeptide chains. Recombinant co-expression of these polypeptides andsubsequent dimerization leads to several possible combinations of thetwo polypeptides. To improve the yield and purity ofprotease-activatable T cell activating bispecific molecules inrecombinant production, it will thus be advantageous to introduce in theFc domain of the protease-activatable T cell activating bispecificmolecule a modification promoting the association of the desiredpolypeptides.

Accordingly, in particular embodiments the Fc domain of theprotease-activatable T cell activating bispecific molecule according tothe invention comprises a modification promoting the association of thefirst and the second subunit of the Fc domain. The site of mostextensive protein-protein interaction between the two subunits of ahuman IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in oneembodiment said modification is in the CH3 domain of the Fc domain.

In a specific embodiment said modification is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g., in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g., tyrosine or tryptophan). Compensatory cavities ofidentical or similar size to the protuberances are created in theinterface of the second polypeptide by replacing large amino acid sidechains with smaller ones (e.g., alanine or threonine). Accordingly, in aparticular embodiment, in the CH3 domain of the first subunit of the Fcdomain of the protease-activatable T cell activating bispecific moleculean amino acid residue is replaced with an amino acid residue having alarger side chain volume, thereby generating a protuberance within theCH3 domain of the first subunit which is positionable in a cavity withinthe CH3 domain of the second subunit, and in the CH3 domain of thesecond subunit of the Fc domain an amino acid residue is replaced withan amino acid residue having a smaller side chain volume, therebygenerating a cavity within the CH3 domain of the second subunit withinwhich the protuberance within the CH3 domain of the first subunit ispositionable.

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g., by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of theFc domain the threonine residue at position 366 is replaced with atryptophan residue (T366W), and in the CH3 domain of the second subunitof the Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V). In one embodiment, in the second subunit of theFc domain additionally the threonine residue at position 366 is replacedwith a serine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesresults in formation of a disulfide bridge between the two subunits ofthe Fc domain, further stabilizing the dimer (Carter, J Immunol Methods248, 7-15 (2001)).

In a particular embodiment the antigen binding moiety capable of bindingto CD3 is fused (optionally via the antigen binding moiety capable ofbinding to a target cell antigen) to the first subunit of the Fc domain(comprising the “knob” modification). Without wishing to be bound bytheory, fusion of the antigen binding moiety capable of binding to CD3to the knob-containing subunit of the Fc domain will (further) minimizethe generation of antigen binding molecules comprising two antigenbinding moieties capable of binding to CD3 (steric clash of twoknob-containing polypeptides).

In an alternative embodiment a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g., as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the protease-activatable T cell activatingbispecific molecule favorable pharmacokinetic properties, including along serum half-life which contributes to good accumulation in thetarget tissue and a favorable tissue-blood distribution ratio. At thesame time it may, however, lead to undesirable targeting of theprotease-activatable T cell activating bispecific molecule to cellsexpressing Fc receptors rather than to the preferred antigen-bearingcells. Moreover, the co-activation of Fc receptor signaling pathways maylead to cytokine release which, in combination with the T cellactivating properties and the long half-life of the antigen bindingmolecule, results in excessive activation of cytokine receptors andsevere side effects upon systemic administration. Activation of (Fcreceptor-bearing) immune cells other than T cells may even reduceefficacy of the protease-activatable T cell activating bispecificmolecule due to the potential destruction of T cells e.g., by NK cells.

Accordingly, in particular embodiments the Fc domain of theprotease-activatable T cell activating bispecific molecules according tothe invention exhibits reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a native IgG₁ Fc domain. Inone such embodiment the Fc domain (or the protease-activatable T cellactivating bispecific molecule comprising said Fc domain) exhibits lessthan 50%, preferably less than 20%, more preferably less than 10% andmost preferably less than 5% of the binding affinity to an Fc receptor,as compared to a native IgG₁ Fc domain (or a protease-activatable T cellactivating bispecific molecule comprising a native IgG₁ Fc domain),and/or less than 50%, preferably less than 20%, more preferably lessthan 10% and most preferably less than 5% of the effector function, ascompared to a native IgG₁ Fc domain domain (or a protease-activatable Tcell activating bispecific molecule comprising a native IgG₁ Fc domain).In one embodiment, the Fc domain domain (or the protease-activatable Tcell activating bispecific molecule comprising said Fc domain) does notsubstantially bind to an Fc receptor and/or induce effector function. Ina particular embodiment the Fc receptor is an Fcγ receptor. In oneembodiment the Fc receptor is a human Fc receptor. In one embodiment theFc receptor is an activating Fc receptor. In a specific embodiment theFc receptor is an activating human Fey receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In oneembodiment the effector function is one or more selected from the groupof CDC, ADCC, ADCP, and cytokine secretion. In a particular embodimentthe effector function is ADCC. In one embodiment the Fc domain domainexhibits substantially similar binding affinity to neonatal Fc receptor(FcRn), as compared to a native IgG₁ Fc domain domain. Substantiallysimilar binding to FcRn is achieved when the Fc domain (or theprotease-activatable T cell activating bispecific molecule comprisingsaid Fc domain) exhibits greater than about 70%, particularly greaterthan about 80%, more particularly greater than about 90% of the bindingaffinity of a native IgG₁ Fc domain (or the protease-activatable T cellactivating bispecific molecule comprising a native IgG₁ Fc domain) toFcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain of the protease-activatable T cell activating bispecificmolecule comprises one or more amino acid mutation that reduces thebinding affinity of the Fc domain to an Fc receptor and/or effectorfunction. Typically, the same one or more amino acid mutation is presentin each of the two subunits of the Fc domain. In one embodiment theamino acid mutation reduces the binding affinity of the Fc domain to anFc receptor. In one embodiment the amino acid mutation reduces thebinding affinity of the Fc domain to an Fc receptor by at least 2-fold,at least 5-fold, or at least 10-fold. In embodiments where there is morethan one amino acid mutation that reduces the binding affinity of the Fcdomain to the Fc receptor, the combination of these amino acid mutationsmay reduce the binding affinity of the Fc domain to an Fc receptor by atleast 10-fold, at least 20-fold, or even at least 50-fold. In oneembodiment the protease-activatable T cell activating bispecificmolecule comprising an engineered Fc domain exhibits less than 20%,particularly less than 10%, more particularly less than 5% of thebinding affinity to an Fc receptor as compared to a protease-activatableT cell activating bispecific molecule comprising a non-engineered Fcdomain. In a particular embodiment the Fc receptor is an Fey receptor.In some embodiments the Fc receptor is a human Fc receptor. In someembodiments the Fc receptor is an activating Fc receptor. In a specificembodiment the Fc receptor is an activating human Fcγ receptor, morespecifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically humanFcγRIIIa. Preferably, binding to each of these receptors is reduced. Insome embodiments binding affinity to a complement component,specifically binding affinity to C1q, is also reduced. In one embodimentbinding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e. preservation of the bindingaffinity of the Fc domain to said receptor, is achieved when the Fcdomain (or the protease-activatable T cell activating bispecificmolecule comprising said Fc domain) exhibits greater than about 70% ofthe binding affinity of a non-engineered form of the Fc domain (or theprotease-activatable T cell activating bispecific molecule comprisingsaid non-engineered form of the Fc domain) to FcRn. The Fc domain, orprotease-activatable T cell activating bispecific molecules of theinvention comprising said Fc domain, may exhibit greater than about 80%and even greater than about 90% of such affinity. In certain embodimentsthe Fc domain of the protease-activatable T cell activating bispecificmolecule is engineered to have reduced effector function, as compared toa non-engineered Fc domain. The reduced effector function can include,but is not limited to, one or more of the following: reduced complementdependent cytotoxicity (CDC), reduced antibody-dependent cell-mediatedcytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis(ADCP), reduced cytokine secretion, reduced immune complex-mediatedantigen uptake by antigen-presenting cells, reduced binding to NK cells,reduced binding to macrophages, reduced binding to monocytes, reducedbinding to polymorphonuclear cells, reduced direct signaling inducingapoptosis, reduced crosslinking of target-bound antibodies, reduceddendritic cell maturation, or reduced T cell priming. In one embodimentthe reduced effector function is one or more selected from the group ofreduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion.In a particular embodiment the reduced effector function is reducedADCC. In one embodiment the reduced ADCC is less than 20% of the ADCCinduced by a non-engineered Fc domain (or a protease-activatable T cellactivating bispecific molecule comprising a non-engineered Fc domain).In one embodiment the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment the Fc domain comprises anamino acid substitution at a position selected from the group of E233,L234, L235, N297, P331 and P329. In a more specific embodiment the Fcdomain comprises an amino acid substitution at a position selected fromthe group of L234, L235 and P329. In some embodiments the Fc domaincomprises the amino acid substitutions L234A and L235A. In one suchembodiment, the Fc domain is an IgG₁ Fc domain, particularly a humanIgG₁ Fc domain. In one embodiment the Fc domain comprises an amino acidsubstitution at position P329. In a more specific embodiment the aminoacid substitution is P329A or P329G, particularly P329G. In oneembodiment the Fc domain comprises an amino acid substitution atposition P329 and a further amino acid substitution at a positionselected from E233, L234, L235, N297 and P331. In a more specificembodiment the further amino acid substitution is E233P, L234A, L235A,L235E, N297A, N297D or P331S. In particular embodiments the Fc domaincomprises amino acid substitutions at positions P329, L234 and L235. Inmore particular embodiments the Fc domain comprises the amino acidmutations L234A, L235A and P329G (“P329G LALA”). In one such embodiment,the Fc domain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain.The “P329G LALA” combination of amino acid substitutions almostcompletely abolishes Fcγ receptor (as well as complement) binding of ahuman IgG₁ Fc domain, as described in PCT publication no. WO2012/130831, incorporated herein by reference in its entirety. WO2012/130831 also describes methods of preparing such mutant Fc domainsand methods for determining its properties such as Fc receptor bindingor effector functions. IgG4 antibodies exhibit reduced binding affinityto Fc receptors and reduced effector functions as compared to IgG₁antibodies. Hence, in some embodiments the Fc domain of theprotease-activatable T cell activating bispecific molecules of theinvention is an IgG4 Fc domain, particularly a human IgG4 Fc domain. Inone embodiment the IgG4 Fc domain comprises amino acid substitutions atposition 5228, specifically the amino acid substitution S228P. Tofurther reduce its binding affinity to an Fc receptor and/or itseffector function, in one embodiment the IgG4 Fc domain comprises anamino acid substitution at position L235, specifically the amino acidsubstitution L235E. In another embodiment, the IgG4 Fc domain comprisesan amino acid substitution at position P329, specifically the amino acidsubstitution P329G. In a particular embodiment, the IgG4 Fc domaincomprises amino acid substitutions at positions 5228, L235 and P329,specifically amino acid substitutions S228P, L235E and P329G. Such IgG4Fc domain mutants and their Fey receptor binding properties aredescribed in PCT publication no. WO 2012/130831, incorporated herein byreference in its entirety.

In a particular embodiment the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG4 Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G.

In certain embodiments N-glycosylation of the Fc domain has beeneliminated. In one such embodiment the Fc domain comprises an amino acidmutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants withsubstitutions at two or more of amino acid positions 265, 269, 270, 297and 327, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g., by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor.

Effector function of an Fc domain, or a protease-activatable T cellactivating bispecific molecule comprising an Fc domain, can be measuredby methods known in the art. A suitable assay for measuring ADCC isdescribed herein. Other examples of in vitro assays to assess ADCCactivity of a molecule of interest are described in U.S. Pat. No.5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986)and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes etal., Proc Natl Acad Sci USA 95, 652-656 (1998). In some embodiments,binding of the Fc domain to a complement component, specifically to C1q,is reduced. Accordingly, in some embodiments wherein the Fc domain isengineered to have reduced effector function, said reduced effectorfunction includes reduced CDC. C1q binding assays may be carried out todetermine whether the protease-activatable T cell activating bispecificmolecule is able to bind C1q and hence has CDC activity. See e.g., C1qand C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assesscomplement activation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al.,Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743(2004)).

Antigen Binding Moieties

The antigen binding molecule of the invention is bispecific, i.e. itcomprises at least two antigen binding moieties capable of specificbinding to two distinct antigenic determinants. According to theinvention, the antigen binding moieties are Fab molecules (i.e. antigenbinding domains composed of a heavy and a light chain, each comprising avariable and a constant region). In one embodiment said Fab moleculesare human. In another embodiment said Fab molecules are humanized. Inyet another embodiment said Fab molecules comprise human heavy and lightchain constant regions.

At least one of the antigen binding moieties is a crossover Fabmolecule. Such modification prevent mispairing of heavy and light chainsfrom different Fab molecules, thereby improving the yield and purity ofthe protease-activatable T cell activating bispecific molecule of theinvention in recombinant production. In a particular crossover Fabmolecule useful for the protease-activatable T cell activatingbispecific molecule of the invention, the constant regions of the Fablight chain and the Fab heavy chain are exchanged. In another crossoverFab molecule useful for the protease-activatable T cell activatingbispecific molecule of the invention, the variable regions of the Fablight chain and the Fab heavy chain are exchanged.

In a particular embodiment according to the invention, theprotease-activatable T cell activating bispecific molecule is capable ofsimultaneous binding to a target cell antigen, particularly a tumor cellantigen, and CD3. In one embodiment, the protease-activatable T cellactivating bispecific molecule is capable of crosslinking a T cell and atarget cell by simultaneous binding to a target cell antigen and CD3. Inan even more particular embodiment, such simultaneous binding results inlysis of the target cell, particularly a tumor cell. In one embodiment,such simultaneous binding results in activation of the T cell. In otherembodiments, such simultaneous binding results in a cellular response ofa T lymphocyte, particularly a cytotoxic T lymphocyte, selected from thegroup of: proliferation, differentiation, cytokine secretion, cytotoxiceffector molecule release, cytotoxic activity, and expression ofactivation markers. In one embodiment, binding of theprotease-activatable T cell activating bispecific molecule to CD3without simultaneous binding to the target cell antigen does not resultin T cell activation.

In one embodiment, the protease-activatable T cell activating bispecificmolecule is capable of re-directing cytotoxic activity of a T cell to atarget cell. In a particular embodiment, said re-direction isindependent of MHC-mediated peptide antigen presentation by the targetcell and and/or specificity of the T cell.

Particularly, a T cell according to any of the embodiments of theinvention is a cytotoxic T cell. In some embodiments the T cell is aCD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

CD3 Binding Moiety

The protease-activatable T cell activating bispecific molecule of theinvention comprises at least one antigen binding moiety capable ofbinding to CD3 (also referred to herein as an “CD3 antigen bindingmoiety” or “first antigen binding moiety”). In a particular embodiment,the protease-activatable T cell activating bispecific molecule comprisesnot more than one antigen binding moiety capable of specific binding toCD3. In one embodiment the protease-activatable T cell activatingbispecific molecule provides monovalent binding to CD3. The CD3 antigenbinding is a crossover Fab molecule, i.e. a Fab molecule wherein eitherthe variable or the constant regions of the Fab heavy and light chainsare exchanged. In embodiments where there is more than one antigenbinding moiety capable of specific binding to a target cell antigencomprised in the protease-activatable T cell activating bispecificmolecule, the antigen binding moiety capable of specific binding to CD3preferably is a crossover Fab molecule and the antigen binding moietiescapable of specific binding to a target cell antigen are conventionalFab molecules.

In a particular embodiment CD3 is human CD3 or cynomolgus CD3, mostparticularly human CD3. In a particular embodiment the CD3 antigenbinding moiety is cross-reactive for (i.e. specifically binds to) humanand cynomolgus CD3. In some embodiments, the first antigen bindingmoiety is capable of specific binding to the epsilon subunit of CD3.

The CD3 antigen binding moiety comprises at least one heavy chaincomplementarity determining region (CDR) selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 and atleast one light chain CDR selected from the group of SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19.

In one embodiment the CD3 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ ID NO: 12, theheavy chain CDR3 of SEQ ID NO: 13, the light chain CDR1 of SEQ ID NO:17, the light chain CDR2 of SEQ ID NO: 18, and the light chain CDR3 ofSEQ ID NO: 19.

In one embodiment the CD3 antigen binding moiety comprises the heavychain CDR1 of SEQ ID NO: 44, the heavy chain CDR2 of SEQ ID NO: 45, theheavy chain CDR3 of SEQ ID NO: 46, the light chain CDR1 of SEQ ID NO:17, the light chain CDR2 of SEQ ID NO: 18, and the light chain CDR3 ofSEQ ID NO: 19.

In one embodiment the CD3 antigen binding moiety comprises a heavy chainvariable region sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 43, and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:55.

In one embodiment the CD3 antigen binding moiety comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 43 anda light chain variable region comprising the amino acid sequence of SEQID NO: 55.

In one embodiment the CD3 antigen binding moiety comprises the heavychain variable region sequence of SEQ ID NO: 43 and the light chainvariable region sequence of SEQ ID NO: 55.

Target Cell Antigen Binding Moiety

The protease-activatable T cell activating bispecific molecule of theinvention comprises at least one antigen binding moiety capable ofbinding to a target cell antigen (also referred to herein as an “targetcell antigen binding moiety” or “second” or “third” antigen bindingmoiety). In certain embodiments, the protease-activatable T cellactivating bispecific molecule comprises two antigen binding moietiescapable of binding to a target cell antigen. In a particular suchembodiment, each of these antigen binding moieties specifically binds tothe same antigenic determinant. In an even more particular embodiment,all of these antigen binding moieties are identical. In one embodiment,the protease-activatable T cell activating bispecific molecule comprisesan immunoglobulin molecule capable of specific binding to a target cellantigen. In one embodiment the protease-activatable T cell activatingbispecific molecule comprises not more than two antigen binding moietiescapable of binding to a target cell antigen.

In a preferred embodiment, the target cell antigen binding moiety is aFab molecule, particularly a conventional Fab molecule that binds to aspecific antigenic determinant and is able to direct theProtease-activatable T cell activating bispecific molecule to a targetsite, for example to a specific type of tumor cell that bears theantigenic determinant.

In certain embodiments the target cell antigen binding moietyspecifically binds to a cell surface antigen. In a particular embodimentthe target cell antigen binding moiety specifically binds to a FolateReceptor 1 (FolR1) on the surface of a target cell. In another specificsuch embodiment the target cell antigen binding moiety specificallybinds to an epidermal growth factor receptor (EGFR), specifically, ahuman EGFR, e.g., HER1. In another specific such embodiment the targetcell antigen binding moiety specifically binds to HER2. In anotherspecific such embodiment the target cell antigen binding moietyspecifically binds to Mesothelin, specifically, to human Mesothelin.

In certain embodiments the target cell antigen binding moiety isdirected to an antigen associated with a pathological condition, such asan antigen presented on a tumor cell or on a virus-infected cell.Suitable antigens are cell surface antigens, for example, but notlimited to, cell surface receptors. In particular embodiments theantigen is a human antigen. In a specific embodiment the target cellantigen is selected from Folate Receptor 1 (FolR1) and epidermal growthfactor receptor (EGFR), specifically, a human EGFR, e.g., HER1. In afurther specific embodiment the target cell antigen is HER2. In afurther specific embodiment the target cell antigen is Mesothelin.

In some embodiments the protease-activatable T cell activatingbispecific molecule comprises at least one antigen binding moiety thatis specific for HER1. In one embodiment, the antigen binding moiety thatis specific for HER1 comprises at least one heavy chain complementaritydetermining region (CDR) of selected from the group consisting of SEQ IDNO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and at least one light chain CDRselected from the group of SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO:61.

In one embodiment, the antigen binding moiety that is specific for HER1comprises the heavy chain CDR1 of SEQ ID NO: 56, the heavy chain CDR2 ofSEQ ID NO: 57, the heavy chain CDR3 of SEQ ID NO: 58, the light chainCDR1 of SEQ ID NO: 59, the light chain CDR2 of SEQ ID NO: 60, and thelight chain CDR3 of SEQ ID NO: 61.

In one embodiment, the antigen binding moiety that is specific for HER1comprises the heavy chain and light chain CDR sequences of an anti-HER1antibody disclosed in PCT Application Publication Number WO2006/082515.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER1 comprises at least one of a polypeptide sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32,a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 33, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 34. In oneembodiment the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER1 comprises the polypeptide sequence of SEQ ID NO: 32, thepolypeptide sequence of SEQ ID NO: 33, and the polypeptide sequence ofSEQ ID NO: 34.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER1 further comprises an anti-idiotypic CD3 scFv comprising atleast one of the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain CDR2of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the light chainCDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, and thelight chain CDR3 of SEQ ID NO: 25. In one embodiment, the anti-idiotypicscFv comprises the heavy chain CDR1 of SEQ ID NO: 20, the heavy chainCDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the lightchain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, andthe light chain CDR3 of SEQ ID NO: 25.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER1 further comprises an anti-idiotypic CD3 scFv comprising atleast one of the heavy chain CDR1 of SEQ ID NO: 26, the heavy chain CDR2of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the light chainCDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID NO: 30, and thelight chain CDR3 of SEQ ID NO: 31. In one embodiment, the anti-idiotypicscFv comprises the heavy chain CDR1 of SEQ ID NO: 26, the heavy chainCDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the lightchain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID NO: 30, andthe light chain CDR3 of SEQ ID NO: 31.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER1 further comprises an anti-idiotypic CD3 scFv comprising apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 41 or 42. In one embodiment, theanti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO: 41or 42.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER1 further comprises an anti-idiotypic HER1 scFv comprising atleast one of the heavy chain CDR1 of SEQ ID NO: 48, the heavy chain CDR2of SEQ ID NO: 49, the heavy chain CDR3 of SEQ ID NO: 50, the light chainCDR1 of SEQ ID NO: 51, the light chain CDR2 of SEQ ID NO: 52, and thelight chain CDR3 of SEQ ID NO: 53. In one embodiment, the anti-idiotypicscFv comprises the heavy chain CDR1 of SEQ ID NO: 48, the heavy chainCDR2 of SEQ ID NO: 49, the heavy chain CDR3 of SEQ ID NO: 50, the lightchain CDR1 of SEQ ID NO: 51, the light chain CDR2 of SEQ ID NO: 52, andthe light chain CDR3 of SEQ ID NO: 53.

In one embodiments the protease-activatable T cell activating bispecificmolecule that comprises at least one antigen binding moiety that isspecific for HER1 further comprises a linker comprising a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 35.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER1 further comprises a linker having a protease recognition sitecomprising a polypeptide sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NO: 36, 37, 38, 39, 40, 97, 98, 99,100, 101, 102, 103, 104, 105 or 106. In one embodiment, the proteaserecognition site comprises the polypeptide sequence of SEQ ID NO: 36,37, 38, 39, 40, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106. In oneembodiment, the protease recognition site comprises the polypeptidesequence of SEQ ID NO: 36. In one embodiment, the protease recognitionsite comprises the polypeptide sequence of SEQ ID NO: 97.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER1 further comprises a linker comprising a polypeptide sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQID NO: 7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In oneembodiment, the linker comprises the polypeptide sequence of SEQ ID NO:7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In oneembodiment, the linker comprises the polypeptide sequence of SEQ ID NO:7. In one embodiment, the linker comprises the polypeptide sequence ofSEQ ID NO: 86.

In some embodiments the protease-activatable T cell activatingbispecific molecule comprises at least one antigen binding moiety thatis specific for HER2. In one embodiment, the antigen binding moiety thatis specific for HER2 comprises at least one heavy chain complementaritydetermining region (CDR) of selected from the group consisting of SEQ IDNO: 142, SEQ ID NO: 143 and SEQ ID NO: 144 and at least one light chainCDR selected from the group of SEQ ID NO: 148, SEQ ID NO: 149, and SEQID NO: 150. In a further one embodiment, the antigen binding moiety thatis specific for HER2 comprises at least one heavy chain complementaritydetermining region (CDR) of selected from the group consisting of SEQ IDNO: 145, SEQ ID NO: 146 and SEQ ID NO: 147 and at least one light chainCDR selected from the group of SEQ ID NO: 148, SEQ ID NO: 149, and SEQID NO: 150.

In one embodiment, the antigen binding moiety that is specific for HER2comprises the heavy chain CDR1 of SEQ ID NO: 142, the heavy chain CDR2of SEQ ID NO: 143, the heavy chain CDR3 of SEQ ID NO: 144, the lightchain CDR1 of SEQ ID NO: 148, the light chain CDR2 of SEQ ID NO: 149,and the light chain CDR3 of SEQ ID NO: 150. In a further embodiment, theantigen binding moiety that is specific for HER2 comprises the heavychain CDR1 of SEQ ID NO: 145, the heavy chain CDR2 of SEQ ID NO: 146,the heavy chain CDR3 of SEQ ID NO: 147, the light chain CDR1 of SEQ IDNO: 148, the light chain CDR2 of SEQ ID NO: 149, and the light chainCDR3 of SEQ ID NO: 150.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER2 further comprises an anti-idiotypic CD3 scFv comprising atleast one of the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain CDR2of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the light chainCDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, and thelight chain CDR3 of SEQ ID NO: 25. In one embodiment, the anti-idiotypicscFv comprises the heavy chain CDR1 of SEQ ID NO: 20, the heavy chainCDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the lightchain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, andthe light chain CDR3 of SEQ ID NO: 25.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER2 further comprises an anti-idiotypic CD3 scFv comprising atleast one of the heavy chain CDR1 of SEQ ID NO: 26, the heavy chain CDR2of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the light chainCDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID NO: 30, and thelight chain CDR3 of SEQ ID NO: 31. In one embodiment, the anti-idiotypicscFv comprises the heavy chain CDR1 of SEQ ID NO: 26, the heavy chainCDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the lightchain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID NO: 30, andthe light chain CDR3 of SEQ ID NO: 31.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER2 further comprises an anti-idiotypic CD3 scFv comprising apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 41 or 42. In one embodiment, theanti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO: 41or 42.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER2 further comprises an anti-idiotypic HER2 scFv comprising atleast one of the heavy chain CDR1 of SEQ ID NO: 48, the heavy chain CDR2of SEQ ID NO: 49, the heavy chain CDR3 of SEQ ID NO: 50, the light chainCDR1 of SEQ ID NO: 51, the light chain CDR2 of SEQ ID NO: 52, and thelight chain CDR3 of SEQ ID NO: 53. In one embodiment, the anti-idiotypicscFv comprises the heavy chain CDR1 of SEQ ID NO: 48, the heavy chainCDR2 of SEQ ID NO: 49, the heavy chain CDR3 of SEQ ID NO: 50, the lightchain CDR1 of SEQ ID NO: 51, the light chain CDR2 of SEQ ID NO: 52, andthe light chain CDR3 of SEQ ID NO: 53.

In one embodiments the protease-activatable T cell activating bispecificmolecule that comprises at least one antigen binding moiety that isspecific for HER2 further comprises a linker comprising a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 35.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER2 further comprises a linker having a protease recognition sitecomprising a polypeptide sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NO: 36, 37, 38, 39, 40, 97, 98, 99,100, 101, 102, 103, 104, 105 or 106. In one embodiment, the proteaserecognition site comprises the polypeptide sequence of SEQ ID NO: 36,37, 38, 39, 40, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106. In oneembodiment, the protease recognition site comprises the polypeptidesequence of SEQ ID NO: 36. In one embodiment, the protease recognitionsite comprises the polypeptide sequence of SEQ ID NO: 97.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor HER2 further comprises a linker comprising a polypeptide sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQID NO: 7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In oneembodiment, the linker comprises the polypeptide sequence of SEQ ID NO:7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In oneembodiment, the linker comprises the polypeptide sequence of SEQ ID NO:7. In one embodiment, the linker comprises the polypeptide sequence ofSEQ ID NO: 86.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 132, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 136, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81 and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 133.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises the polypeptide sequence of SEQ ID NO: 132, thepolypeptide sequence of SEQ ID NO: 136, the polypeptide sequence of SEQID NO: 81 and the polypeptide sequence of SEQ ID NO: 133.

In particular embodiments the protease-activatable T cell activatingbispecific molecule comprises at least one antigen binding moiety thatis specific for FolR1. In one embodiment the FolR1 is a human FolR1. Inone embodiment, the protease-activatable T cell activating bispecificmolecule comprises at least one antigen binding moiety that is specificfor human FolR1 and does not bind to human FolR2 or human FolR3. In oneembodiment, the antigen binding moiety that is specific for FolR1comprises at least one heavy chain complementarity determining region(CDR) selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15and SEQ ID NO: 16 and at least one light chain CDR selected from thegroup of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

In one embodiment, the antigen binding moiety that is specific for FolR1comprises the heavy chain CDR1 of SEQ ID NO: 14, the heavy chain CDR2 ofSEQ ID NO: 15, the heavy chain CDR3 of SEQ ID NO: 16, the light chainCDR1 of SEQ ID NO: 17, the light chain CDR2 of SEQ ID NO: 18, and thelight chain CDR3 of SEQ ID NO: 19.

In a further embodiment, the antigen binding moiety that is specific forFolR1 comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 55, or variants thereofthat retain functionality.

In one embodiment, the antigen binding moiety that is specific for FolR1comprises the heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 47 and the light chain variable region comprisingan amino acid sequence of SEQ ID NO: 55.

In one embodiment, the antigen binding moiety that is specific for FolR1comprises at least one heavy chain complementarity determining region(CDR) selected from the group consisting of SEQ ID NO: 151, SEQ ID NO:152 and SEQ ID NO: 153 and at least one light chain CDR selected fromthe group of SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156.

In one embodiment, the antigen binding moiety that is specific for FolR1comprises the heavy chain CDR1 of SEQ ID NO: 151, the heavy chain CDR2of SEQ ID NO: 152, the heavy chain CDR3 of SEQ ID NO: 153, the lightchain CDR1 of SEQ ID NO: 154, the light chain CDR2 of SEQ ID NO: 155,and the light chain CDR3 of SEQ ID NO: 156.

In a further embodiment, the antigen binding moiety that is specific forFolR1 comprises a heavy chain variable region sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 157 and alight chain variable region sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 158, or variants thereofthat retain functionality.

In one embodiment, the antigen binding moiety that is specific for FolR1comprises the heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 157 and the light chain variable regioncomprising an amino acid sequence of SEQ ID NO: 158.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, and a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 1.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises the polypeptide sequence of SEQ ID NO: 2, and thepolypeptide sequence of SEQ ID NO: 1.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor FolR1 further comprises an anti-idiotypic CD3 scFv comprising atleast one of the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain CDR2of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the light chainCDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, and thelight chain CDR3 of SEQ ID NO: 25. In one embodiment, the anti-idiotypicscFv comprises the heavy chain CDR1 of SEQ ID NO: 20, the heavy chainCDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the lightchain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, andthe light chain CDR3 of SEQ ID NO: 25.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor FolR1 further comprises an anti-idiotypic CD3 scFv comprising atleast one of the heavy chain CDR1 of SEQ ID NO: 26, the heavy chain CDR2of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the light chainCDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID NO: 30, and thelight chain CDR3 of SEQ ID NO: 31. In one embodiment, the anti-idiotypicscFv comprises the heavy chain CDR1 of SEQ ID NO: 26, the heavy chainCDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the lightchain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID NO: 30, andthe light chain CDR3 of SEQ ID NO: 31.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor FolR1 further comprises an anti-idiotypic CD3 scFv comprising apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 41 or 42. In one embodiment, theanti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO: 41or 42.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor FolR1 further comprises a linker having a protease recognition sitecomprising a polypeptide sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NO: 36, 37, 38, 39, 40, 97, 98, 99,100, 101, 102, 103, 104, 105 or 106. In one embodiment, the proteaserecognition site comprises the polypeptide sequence of SEQ ID NO: 36,37, 38, 39, 40, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106. In oneembodiment, the protease recognition site comprises the polypeptidesequence of SEQ ID NO: 36. In one embodiment, the protease recognitionsite comprises the polypeptide sequence of SEQ ID NO: 97. In oneembodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor ForR1 further comprises a linker comprising a polypeptide sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQID NO: 7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In oneembodiment, the linker comprises the polypeptide sequence of SEQ ID NO:7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In oneembodiment, the linker comprises the polypeptide sequence of SEQ ID NO:7. In one embodiment, the linker comprises the polypeptide sequence ofSEQ ID NO: 86.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 3 and a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 72.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 3 and a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 72.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 3 and a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 85.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 3, a polypeptide sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 73 and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 74.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises the polypeptide sequence of SEQ ID NO: 1, thepolypeptide sequence of SEQ ID NO: 3 and the polypeptide sequence of SEQID NO: 72.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises the polypeptide sequence of SEQ ID NO: 1, thepolypeptide sequence of SEQ ID NO: 3 and the polypeptide sequence of SEQID NO: 85.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises the polypeptide sequence of SEQ ID NO: 1, thepolypeptide sequence of SEQ ID NO: 3, the polypeptide sequence of SEQ IDNO: 73 and the polypeptide sequence of SEQ ID NO: 74.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 137, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 139, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81 and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 138.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises the polypeptide sequence of SEQ ID NO: 137, thepolypeptide sequence of SEQ ID NO: 139, the polypeptide sequence of SEQID NO: 81 and the polypeptide sequence of SEQ ID NO: 138.

In particular embodiments the protease-activatable T cell activatingbispecific molecule comprises at least one antigen binding moiety thatis specific for Mesothelin. In one embodiment the Mesothelin is humanMesothelin. In one embodiment, the protease-activatable T cellactivating bispecific molecule comprises at least one antigen bindingmoiety that is specific for human Mesothelin. In one embodiment, theantigen binding moiety that is specific for Mesothelin comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ IDNO: 109 and at least one light chain CDR selected from the group of SEQID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112.

In one embodiment, the antigen binding moiety that is specific forMesothelin comprises the heavy chain CDR1 of SEQ ID NO: 107, the heavychain CDR2 of SEQ ID NO: 108, the heavy chain CDR3 of SEQ ID NO: 109,the light chain CDR1 of SEQ ID NO: 110, the light chain CDR2 of SEQ IDNO: 111, and the light chain CDR3 of SEQ ID NO: 112.

In a further embodiment, the antigen binding moiety that is specific forMesothelin comprises a heavy chain variable region sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 113and a light chain variable region sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 114, or variantsthereof that retain functionality.

In one embodiment, the antigen binding moiety that is specific forMesothelin comprises the heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 113 and the light chain variableregion comprising the amino acid sequence of SEQ ID NO: 114.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor Mesothelin further comprises an anti-idiotypic CD3 scFv comprisingat least one of the heavy chain CDR1 of SEQ ID NO: 20, the heavy chainCDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the lightchain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, andthe light chain CDR3 of SEQ ID NO: 25. In one embodiment, theanti-idiotypic scFv comprises the heavy chain CDR1 of SEQ ID NO: 20, theheavy chain CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO:22, the light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQID NO: 24, and the light chain CDR3 of SEQ ID NO: 25.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor Mesothelin further comprises an anti-idiotypic CD3 scFv comprisingat least one of the heavy chain CDR1 of SEQ ID NO: 26, the heavy chainCDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the lightchain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID NO: 30, andthe light chain CDR3 of SEQ ID NO: 31. In one embodiment, theanti-idiotypic scFv comprises the heavy chain CDR1 of SEQ ID NO: 26, theheavy chain CDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO:28, the light chain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQID NO: 30, and the light chain CDR3 of SEQ ID NO: 31.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor Mesothelin further comprises an anti-idiotypic CD3 scFv comprising apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 41 or 42. In one embodiment, theanti-idiotypic scFv comprises the polypeptide sequence of SEQ ID NO: 41or 42.

In one embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor Mesothelin further comprises a linker having a protease recognitionsite comprising a polypeptide sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 36, 37, 38, 39, 40, 97,98, 99, 100, 101, 102, 103, 104, 105 or 106. In one embodiment, theprotease recognition site comprises the polypeptide sequence of SEQ IDNO: 36, 37, 38, 39, 40, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106.In one embodiment, the protease recognition site comprises thepolypeptide sequence of SEQ ID NO: 36. In one embodiment, the proteaserecognition site comprises the polypeptide sequence of SEQ ID NO: 97. Inone embodiments the protease-activatable T cell activating bispecificmolecule comprising at least one antigen binding moiety that is specificfor Mesothelin further comprises a linker comprising a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94,95 or 96. In one embodiment, the linker comprises the polypeptidesequence of SEQ ID NO: 7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94,95 or 96. In one embodiment, the linker comprises the polypeptidesequence of SEQ ID NO: 7. In one embodiment, the linker comprises thepolypeptide sequence of SEQ ID NO: 86.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 77, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 78, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81 and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 82.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises a polypeptide sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 76, a polypeptidesequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 77, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 78 and apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 79.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises the polypeptide sequence of SEQ ID NO: 77, thepolypeptide sequence of SEQ ID NO: 78, the polypeptide sequence of SEQID NO: 81 and the polypeptide sequence of SEQ ID NO: 82.

In one embodiment the protease-activatable T cell activating bispecificmolecule comprises the polypeptide sequence of SEQ ID NO: 76, thepolypeptide sequence of SEQ ID NO: 77, the polypeptide sequence of SEQID NO: 78 and the polypeptide sequence of SEQ ID NO: 79.

In one embodiment, provided is a T cell activating bispecific moleculecomprises a polypeptide sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NO: 76, a polypeptide sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNO: 77, a polypeptide sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NO: 78 and a polypeptide sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQID NO: 81.

In one embodiment the T cell activating bispecific molecule comprises apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 77, a polypeptide sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 78, apolypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 81 and a polypeptide sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 84.

In one embodiment the T cell activating bispecific molecule comprisesthe polypeptide sequence of SEQ ID NO: 76, the polypeptide sequence ofSEQ ID NO: 77, the polypeptide sequence of SEQ ID NO: 78 and thepolypeptide sequence of SEQ ID NO: 81.

In one embodiment the T cell activating bispecific molecule comprisesthe polypeptide sequence of SEQ ID NO: 77, the polypeptide sequence ofSEQ ID NO: 78, the polypeptide sequence of SEQ ID NO: 81 and thepolypeptide sequence of SEQ ID NO: 84.

Masking Moiety

The protease-activatable T cell activating bispecific molecule of theinvention comprises at least one masking moiety. Others have tried tomask binding of an antibody by capping the binding moiety with afragment of the antigen recognized by the binding moiety (e.g.,WO2013128194). This approach has several limitations. For example, usingthe antigen allows for less flexibility in reducing the affinity of thebinding moiety. This is so because the affinity has to be high enough tobe reliably masked by the antigen mask. Also, dissociated antigen couldpotentially bind to and interact with its cognate receptor(s) in vivoand cause undesirable signals to the cell expressing such receptor. Incontrast, the approach described herein uses an anti-idiotype antibodyor fragment thereof as a mask. Two countervailing considerations fordesigning an effective masking moiety are 1. effectiveness of themasking and 2. reversibility of the masking. If the affinity is too low,masking would be inefficient. However, if the affinity is too high, themasking process might not be readily reversible. It was not predictablewhether a high affinity anti-idiotype mask or a low affinityanti-idiotype mask would work better. As described herein, higheraffinity masking moieties performed overall better in masking theantigen binding side and, at the same time, could be effectively removedfor activation of the molecule. In one embodiment, the anti-idiotypemask has a KD of 1-8 nM. In one embodiment, anti-idiotype mask has a KDof 2 nM at 37° C. In one specific embodiment, the masking moietyrecognizes the idiotype of the first antigen binding moiety capable ofspecific binding to a CD3, e.g., a human CD3. In one specificembodiment, the masking moiety recognizes the idiotype of the secondantigen binding moiety capable of binding to a target cell antigen.

In one embodiment, the masking moiety masks a CD3-binding moiety andcomprises at least one of the heavy chain CDR1 of SEQ ID NO: 20, theheavy chain CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO:22, the light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQID NO: 24, and the light chain CDR3 of SEQ ID NO: 25. In one embodiment,the masking moiety comprises the heavy chain CDR1 of SEQ ID NO: 20, theheavy chain CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO:22, the light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQID NO: 24, and the light chain CDR3 of SEQ ID NO: 25.

In one embodiment, the masking moiety masks a CD3-binding moiety andcomprises a polypeptide sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NO: 41. In one embodiment, themasking moiety masks a CD3-binding moiety and comprises the polypeptidesequence of SEQ ID NO: 41.

In one preferred embodiment, the masking moiety masks a CD3-bindingmoiety and comprises at least one of the heavy chain CDR1 of SEQ ID NO:26, the heavy chain CDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQID NO: 28, the light chain CDR1 of SEQ ID NO: 29, the light chain CDR2of SEQ ID NO: 30, and the light chain CDR3 of SEQ ID NO: 31. In oneembodiment, the masking moiety comprises the heavy chain CDR1 of SEQ IDNO: 26, the heavy chain CDR2 of SEQ ID NO: 27, the heavy chain CDR3 ofSEQ ID NO: 28, the light chain CDR1 of SEQ ID NO: 29, the light chainCDR2 of SEQ ID NO: 30, and the light chain CDR3 of SEQ ID NO: 31. In oneembodiment, the masking moiety masks a CD3-binding moiety and comprisesa polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO: 42. In one preferred embodiment, themasking moiety masks a CD3-binding moiety and comprises the polypeptidesequence of SEQ ID NO: 42.

In one embodiment, the masking moiety masks a HER1-binding moiety andcomprises at least one of the heavy chain CDR1 of SEQ ID NO: 48, theheavy chain CDR2 of SEQ ID NO: 49, the heavy chain CDR3 of SEQ ID NO:50, the light chain CDR1 of SEQ ID NO: 51, the light chain CDR2 of SEQID NO: 52, and the light chain CDR3 of SEQ ID NO: 53. In one embodiment,the anti-idiotypic scFv comprises the heavy chain CDR1 of SEQ ID NO: 48,the heavy chain CDR2 of SEQ ID NO: 49, the heavy chain CDR3 of SEQ IDNO: 50, the light chain CDR1 of SEQ ID NO: 51, the light chain CDR2 ofSEQ ID NO: 52, and the light chain CDR3 of SEQ ID NO: 53.

In one aspect, the invention relates to an idiotype-specific polypeptidefor reversibly concealing antigen binding of an antigen-binding of amolecule. In one embodiment, the invention relates to anidiotype-specific polypeptide for reversibly concealing an anti-CD3antigen binding site of a molecule. Such idiotype-specific polypeptidefor reversibly concealing an anti-CD3 antigen binding site must becapable of specific binding to the anti-CD3 antigen binding site'sidiotype and thereby reducing or abrogating binding of the anti-CD3antigen binding site to CD3. In one embodiment, the invention relates toan idiotype-specific polypeptide for reversibly concealing an anti-HER1antigen binding site of a molecule. In one embodiment theidiotype-specific polypeptide is an anti-idiotype scFv. In oneembodiment the idiotype-specific polypeptide is covalently attached tothe molecule through a linker. In one embodiment the idiotype-specificpolypeptide is covalently attached to the molecule through more than onelinker. In one embodiment the idiotype-specific polypeptide iscovalently attached to the molecule through two linkers. In oneembodiment the linker is a peptide linker. In one embodiment the linkeris a protease-cleavable linker. In one embodiment, the linker comprisesthe sequence of SEQ ID NO: 7, 8, 9, or 10. In one embodiment, the linkercomprises the sequence of SEQ ID NO: 7, 8, 9, 10, 86, 87, 88, 89, 90,91, 92, 93, 94, 95 or 96. In one embodiment, the linker comprises thepolypeptide sequence of SEQ ID NO: 7. In one embodiment, the linkercomprises the polypeptide sequence of SEQ ID NO: 86. In one embodimentthe peptide linker comprises at least one protease recognition site. Inone embodiment, the protease recognition site comprises the polypeptidesequence of SEQ ID NO: 36, 37, 38, 39, 40, 97, 98, 99, 100, 101, 102,103, 104, 105 or 106. In one preferred embodiment, the proteaserecognition site comprises the protease recognition sequence RQARVVNG(SEQ ID NO: 36). In further embodiment, the linker comprises more thanone protease recognition site. In one preferred embodiment, the proteaserecognition site comprises the protease recognition sequenceVHMPLGFLGPRQARVVNG (SEQ ID NO:97). In one embodiment the protease isselected from the group consisting of metalloproteinase, e.g., matrixmetalloproteinase (MMP) 1-28 and A Disintegrin And Metalloproteinase(ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serine protease, e.g.,urokinase-type plasminogen activator and Matriptase, cysteine protease,aspartic protease, and cathepsin protease. In one specific embodimentthe protease is MMP9 or MMP2. In a further specific embodiment, theprotease is Matriptase.

In one embodiment the molecule which comprises the anti-CD3 antigenbinding site is a T-cell activating bispecific molecule. In oneparticular embodiment the idiotype-specific polypeptide comprises aheavy chain variable region comprising at least one of a heavy chaincomplementarity determining region (CDR H) 1 amino acid sequence ofDYSIH (SEQ ID NO:20); CDR H2 amino acid sequence of WINTETGEPAYADDFKG(SEQ ID NO:21); and a CDR H3 amino acid sequence of PYDYDVLDY (SEQ IDNO:22). In one particular embodiment the idiotype-specific polypeptidecomprises a light chain variable region comprising at least one of: alight chain (CDR L)1 amino acid sequence of RASKSVSTSNYSYIH (SEQ IDNO:23); a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24); and aCDR L3 amino acid sequence of QHSREFPWT (SEQ ID NO:25). In oneparticular embodiment the idiotype-specific polypeptide comprises: aheavy chain complementarity determining region (CDR H) 1 amino acidsequence of DYSIH (SEQ ID NO:20); a CDR H2 amino acid sequence ofWINTETGEPAYADDFKG (SEQ ID NO:21); a CDR H3 amino acid sequence ofPYDYDVLDY (SEQ ID NO:22); and a light chain variable region comprising:a light chain (CDR L)1 amino acid sequence of RASKSVSTSNYSYIH (SEQ IDNO:23); a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24); and aCDR L3 amino acid sequence of QHSREFPWT (SEQ ID NO:25). In oneparticular embodiment the idiotype-specific polypeptide comprises aheavy chain variable region comprising at least one of: a heavy chaincomplementarity determining region (CDR H) 1 amino acid sequence ofSYGVS (SEQ ID NO:26); a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS(SEQ ID NO:27); and a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQID NO:28). In one particular embodiment the idiotype-specificpolypeptide comprises a light chain variable region comprising at leastone of: a light chain (CDR L)1 amino acid sequence of RASENIDSYLA (SEQID NO:29); a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30); and aCDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO:31). In oneparticular embodiment the idiotype-specific polypeptide comprises aheavy chain variable region comprising: a heavy chain complementaritydetermining region (CDR H) 1 amino acid sequence of SYGVS (SEQ IDNO:26); a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID NO:27);a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO:28); and alight chain variable region comprising: a light chain (CDR L)1 aminoacid sequence of RASENIDSYLA (SEQ ID NO:29); a CDR L2 amino acidsequence of AATFLAD (SEQ ID NO:30); and a CDR L3 amino acid sequence ofQHYYSTPYT (SEQ ID NO:31). In one embodiment, the idiotype-specificpolypeptide comprises a heavy chain variable region comprising at leastone of: a heavy chain complementarity determining region (CDR H) 1 aminoacid sequence of SEQ ID NO:48; a CDR H2 amino acid sequence of SEQ IDNO:49; and a CDR H3 amino acid sequence of SEQ ID NO:50. In oneembodiment, the idiotype-specific polypeptide comprises a light chainvariable region comprising at least one of: a light chaincomplementarity determining region (CDR L) 1 amino acid sequence of SEQID NO:51; a CDR L2 amino acid sequence of SEQ ID NO:52; and a CDR L3amino acid sequence of SEQ ID NO:53. In one embodiment, theidiotype-specific polypeptide comprises a heavy chain variable regioncomprising a heavy chain complementarity determining region (CDR H) 1amino acid sequence of SEQ ID NO:48; a CDR H2 amino acid sequence of SEQID NO:49; and a CDR H3 amino acid sequence of SEQ ID NO:50, and a lightchain variable region comprising a light chain complementaritydetermining region (CDR L) 1 amino acid sequence of SEQ ID NO:51; a CDRL2 amino acid sequence of SEQ ID NO:52; and a CDR L3 amino acid sequenceof SEQ ID NO:53.

Polynucleotides

The invention further provides isolated polynucleotides encoding aprotease-activatable T cell activating bispecific molecule as describedherein or a fragment thereof. In some embodiments, said fragment is anantigen binding fragment.

Polynucleotides of the invention include those that are at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequences set forth in SEQ ID NOs 62-71 or SEQ ID NOs includingfunctional fragments or variants thereof. Polynucleotides of theinvention further include those that are at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set forth inSEQ ID NOs 117-131 including functional fragments or variants thereof.Polynucleotides of the invention further include those that are at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequences set forth in SEQ ID NOs 162-170 including functional fragmentsor variants thereof.

The polynucleotides encoding protease-activatable T cell activatingbispecific molecules of the invention may be expressed as a singlepolynucleotide that encodes the entire protease-activatable T cellactivating bispecific molecule or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional protease-activatableT cell activating bispecific molecule. For example, the light chainportion of an antigen binding moiety may be encoded by a separatepolynucleotide from the portion of the protease-activatable T cellactivating bispecific molecule comprising the heavy chain portion of theantigen binding moiety, an Fc domain subunit and optionally (part of)another antigen binding moiety. When co-expressed, the heavy chainpolypeptides will associate with the light chain polypeptides to formthe antigen binding moiety. In another example, the portion of theprotease-activatable T cell activating bispecific molecule comprisingone of the two Fc domain subunits and optionally (part of) one or moreantigen binding moieties could be encoded by a separate polynucleotidefrom the portion of the protease-activatable T cell activatingbispecific molecule comprising the the other of the two Fc domainsubunits and optionally (part of) an antigen binding moiety. Whenco-expressed, the Fc domain subunits will associate to form the Fcdomain.

In some embodiments, the isolated polynucleotide encodes the entireprotease-activatable T cell activating bispecific molecule according tothe invention as described herein. In other embodiments, the isolatedpolynucleotide encodes a polypeptides comprised in theprotease-activatable T cell activating bispecific molecule according tothe invention as described herein.

In another embodiment, the present invention is directed to an isolatedpolynucleotide encoding a protease-activatable T cell activatingbispecific molecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence. In another embodiment, the present invention is directed to anisolated polynucleotide encoding a protease-activatable T cellactivating bispecific molecule or fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a polypeptide sequenceas shown in SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 47, or 55. In another embodiment, thepresent invention is directed to an isolated polynucleotide encoding aprotease-activatable T cell activating bispecific molecule or fragmentthereof, wherein the polynucleotide comprises a sequence that encodes apolypeptide sequence as shown in SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 47, 55, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85. In another embodiment, thepresent invention is directed to an isolated polynucleotide encoding aprotease-activatable T cell activating bispecific molecule or fragmentthereof, wherein the polynucleotide comprises a sequence that encodes apolypeptide sequence as shown in SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 47, 55, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 132, 133, 134, 135, 136,137, 138, 139, 140 or 141. In another embodiment, the invention isfurther directed to an isolated polynucleotide encoding aprotease-activatable T cell activating bispecific molecule of theinvention or a fragment thereof, wherein the polynucleotide comprises asequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to the nucleotide sequence shown in SEQ ID NOs 62, 63, 64,65, 66, 67, 68, 69, 70, or 71. In another embodiment, the invention isfurther directed to an isolated polynucleotide encoding aprotease-activatable T cell activating bispecific molecule of theinvention or a fragment thereof, wherein the polynucleotide comprises asequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to the nucleotide sequence shown in SEQ ID NOs 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130 or 131. In another embodiment, the invention isfurther directed to an isolated polynucleotide encoding aprotease-activatable T cell activating bispecific molecule of theinvention or a fragment thereof, wherein the polynucleotide comprises asequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to the nucleotide sequence shown in SEQ ID NOs 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 162, 163, 164, 165, 166, 167, 168, 169 or170. In another embodiment, the invention is directed to an isolatedpolynucleotide encoding a protease-activatable T cell activatingbispecific molecule of the invention or a fragment thereof, wherein thepolynucleotide comprises the nucleic acid sequence shown in SEQ ID NOs62, 63, 64, 65, 66, 67, 68, 69, 70, or 71. In another embodiment, theinvention is directed to an isolated polynucleotide encoding aprotease-activatable T cell activating bispecific molecule of theinvention or a fragment thereof, wherein the polynucleotide comprisesthe nucleic acid sequence shown in SEQ ID NOs 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130 or 131. In another embodiment, the invention is directedto an isolated polynucleotide encoding a protease-activatable T cellactivating bispecific molecule of the invention or a fragment thereof,wherein the polynucleotide comprises the nucleic acid sequence shown inSEQ ID NOs 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 162, 163, 164,165, 166, 167, 168, 169 or 170. In another embodiment, the invention isdirected to an isolated polynucleotide encoding a protease-activatable Tcell activating bispecific molecule of the invention or a fragmentthereof, wherein the polynucleotide comprises a sequence that encodes avariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence in SEQ ID NOs 43,47, or 55. The invention encompasses an isolated polynucleotide encodinga protease-activatable T cell activating bispecific molecule of theinvention or a fragment thereof, wherein the polynucleotide comprises asequence that encodes the variable region sequence of SEQ ID NOs SEQ IDNOs 43, 47, or 55 with conservative amino acid substitutions.

In another embodiment, the invention is directed to an isolatedpolynucleotide encoding a protease-activatable T cell activatingbispecific molecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to the amino acid sequence in SEQ ID NOs 43, 47, 55, 113,114, 115 or 116. The invention encompasses an isolated polynucleotideencoding a protease-activatable T cell activating bispecific molecule ofthe invention or a fragment thereof, wherein the polynucleotidecomprises a sequence that encodes the variable region sequence of SEQ IDNOs SEQ ID NOs 43, 47, 55, 113, 114, 115 or 116 with conservative aminoacid substitutions.

In another embodiment, the invention is directed to an isolatedpolynucleotide encoding a protease-activatable T cell activatingbispecific molecule of the invention or a fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to the amino acid sequence in SEQ ID NOs 43, 47, 55, 113,114, 115, 116, 157, 158, 159, 160 or 161. The invention encompasses anisolated polynucleotide encoding a protease-activatable T cellactivating bispecific molecule of the invention or a fragment thereof,wherein the polynucleotide comprises a sequence that encodes thevariable region sequence of SEQ ID NOs SEQ ID NOs 43, 47, 55, 113, 114,115, 116, 157, 158, 159, 160 or 161 with conservative amino acidsubstitutions.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

The invention further provides isolated polynucleotides encoding anidiotype-specific polypeptide as described herein or a fragment thereof.In some embodiments, said fragment is an idiotype binding, i.e.,anti-idiotype specific antibody or fragment thereof. In one embodimentthe idiotype-specific polypeptide is an anti-idiotypic scFv.

The invention also encompasses an isolated polynucleotide encoding anidiotype-specific polypeptide of the invention or a fragment thereof,wherein the polynucleotide comprises a sequence that encodes thepolypeptide sequence of one or more of SEQ ID NOs 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 48, 49, 50, 51, 52, and 53. The inventionalso encompasses an isolated polynucleotide encoding anidiotype-specific polypeptide of the invention or a fragment thereof,wherein the polynucleotide comprises a sequence that encodes thepolypeptide sequence of one or more of SEQ ID NOs 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 48, 49, 50, 51, 52, and 53 with conservativeamino acid substitutions.

The polynucleotides encoding idiotype-specific polypeptides of theinvention may be expressed as a single polynucleotide that encodes theentire idiotype-specific polypeptide or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional idiotype-specificpolypeptide, e.g., a masking moiety. For example, in one embodiment theidiotype-specific polypeptide is an anti-idiotypic scFv (single chainvariable fragment) wherein the light chain variable portion of theanti-idiotypic scFv may be encoded by a separate polynucleotide from theportion of the anti-idiotypic scFv comprising the heavy chain variableportion of the anti-idiotypic scFv. When co-expressed, the heavy chainpolypeptides will associate with the light chain polypeptides to formthe anti-idiotypic scFv. In some embodiments, the isolatedpolynucleotide encodes the idiotype-specific polypeptide according tothe invention as described herein. In certain embodiments thepolynucleotide or nucleic acid is DNA. In other embodiments, apolynucleotide of the present invention is RNA, for example, in the formof messenger RNA (mRNA). RNA of the present invention may be singlestranded or double stranded.

Recombinant Methods

protease-activatable T cell activating bispecific molecules of theinvention may be obtained, for example, by solid-state peptide synthesis(e.g., Merrifield solid phase synthesis) or recombinant production. Forrecombinant production one or more polynucleotide encoding theprotease-activatable T cell activating bispecific molecule (fragment),e.g., as described above, is isolated and inserted into one or morevectors for further cloning and/or expression in a host cell. Suchpolynucleotide may be readily isolated and sequenced using conventionalprocedures. In one embodiment a vector, preferably an expression vector,comprising one or more of the polynucleotides of the invention isprovided. Methods which are well known to those skilled in the art canbe used to construct expression vectors containing the coding sequenceof a protease-activatable T cell activating bispecific molecule(fragment) along with appropriate transcriptional/translational controlsignals. These methods include in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.See, for example, the techniques described in Maniatis et al., MOLECULARCLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y.(1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,Greene Publishing Associates and Wiley Interscience, N.Y (1989). Theexpression vector can be part of a plasmid, virus, or may be a nucleicacid fragment. The expression vector includes an expression cassetteinto which the polynucleotide encoding the protease-activatable T cellactivating bispecific molecule (fragment) (i.e. the coding region) iscloned in operable association with a promoter and/or othertranscription or translation control elements. As used herein, a “codingregion” is a portion of nucleic acid which consists of codons translatedinto amino acids. Although a “stop codon” (TAG, TGA, or TAA) is nottranslated into an amino acid, it may be considered to be part of acoding region, if present, but any flanking sequences, for examplepromoters, ribosome binding sites, transcriptional terminators, introns,5′ and 3′ untranslated regions, and the like, are not part of a codingregion. Two or more coding regions can be present in a singlepolynucleotide construct, e.g., on a single vector, or in separatepolynucleotide constructs, e.g., on separate (different) vectors.Furthermore, any vector may contain a single coding region, or maycomprise two or more coding regions, e.g., a vector of the presentinvention may encode one or more polypeptides, which are post- orco-translationally separated into the final proteins via proteolyticcleavage. In addition, a vector, polynucleotide, or nucleic acid of theinvention may encode heterologous coding regions, either fused orunfused to a polynucleotide encoding the protease-activatable T cellactivating bispecific molecule (fragment) of the invention, or variantor derivative thereof. Heterologous coding regions include withoutlimitation specialized elements or motifs, such as a secretory signalpeptide or a heterologous functional domain. An operable association iswhen a coding region for a gene product, e.g., a polypeptide, isassociated with one or more regulatory sequences in such a way as toplace expression of the gene product under the influence or control ofthe regulatory sequence(s). Two DNA fragments (such as a polypeptidecoding region and a promoter associated therewith) are “operablyassociated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein. A variety of transcription controlregions are known to those skilled in the art. These include, withoutlimitation, transcription control regions, which function in vertebratecells, such as, but not limited to, promoter and enhancer segments fromcytomegaloviruses (e.g., the immediate early promoter, in conjunctionwith intron-A), simian virus 40 (e.g., the early promoter), andretroviruses (such as, e.g., Rous sarcoma virus). Other transcriptioncontrol regions include those derived from vertebrate genes such asactin, heat shock protein, bovine growth hormone and rabbit â-globin, aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control regionsinclude tissue-specific promoters and enhancers as well as induciblepromoters (e.g., promoters inducible tetracyclins). Similarly, a varietyof translation control elements are known to those of ordinary skill inthe art. These include, but are not limited to ribosome binding sites,translation initiation and termination codons, and elements derived fromviral systems (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). The expression cassette may alsoinclude other features such as an origin of replication, and/orchromosome integration elements such as retroviral long terminal repeats(LTRs), or adeno-associated viral (AAV) inverted terminal repeats(ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the protease-activatable T cell activating bispecific molecule isdesired, DNA encoding a signal sequence may be placed upstream of thenucleic acid encoding a protease-activatable T cell activatingbispecific molecule of the invention or a fragment thereof. According tothe signal hypothesis, proteins secreted by mammalian cells have asignal peptide or secretory leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Those of ordinary skill in theart are aware that polypeptides secreted by vertebrate cells generallyhave a signal peptide fused to the N-terminus of the polypeptide, whichis cleaved from the translated polypeptide to produce a secreted or“mature” form of the polypeptide. In certain embodiments, the nativesignal peptide, e.g., an immunoglobulin heavy chain or light chainsignal peptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, may be used. Forexample, the wild-type leader sequence may be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g., a histidine tag) or assist in labeling theprotease-activatable T cell activating bispecific molecule may beincluded within or at the ends of the protease-activatable T cellactivating bispecific molecule (fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g., has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) aprotease-activatable T cell activating bispecific molecule of theinvention. As used herein, the term “host cell” refers to any kind ofcellular system which can be engineered to generate theprotease-activatable T cell activating bispecific molecules of theinvention or fragments thereof. Host cells suitable for replicating andfor supporting expression of protease-activatable T cell activatingbispecific molecules are well known in the art. Such cells may betransfected or transduced as appropriate with the particular expressionvector and large quantities of vector containing cells can be grown forseeding large scale fermenters to obtain sufficient quantities of theprotease-activatable T cell activating bispecific molecule for clinicalapplications. Suitable host cells include prokaryotic microorganisms,such as E. coli, or various eukaryotic cells, such as Chinese hamsterovary cells (CHO), insect cells, or the like. For example, polypeptidesmay be produced in bacteria in particular when glycosylation is notneeded. After expression, the polypeptide may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forpolypeptide-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized”, resulting in theproduction of a polypeptide with a partially or fully humanglycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004),and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells forthe expression of (glycosylated) polypeptides are also derived frommulticellular organisms (invertebrates and vertebrates). Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains have been identified which may be used in conjunction withinsect cells, particularly for transfection of Spodoptera frugiperdacells. Plant cell cultures can also be utilized as hosts. See e.g., U.S.Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants). Vertebrate cells may also be used as hosts. Forexample, mammalian cell lines that are adapted to grow in suspension maybe useful. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line(293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36,59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)),monkey kidney cells (CV1), African green monkey kidney cells (VERO-76),human cervical carcinoma cells (HELA), canine kidney cells (MDCK),buffalo rat liver cells (BRL 3A), human lung cells (W138), human livercells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (asdescribed, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68(1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host celllines include Chinese hamster ovary (CHO) cells, including dhfr⁻ CHOcells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); andmyeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review ofcertain mammalian host cell lines suitable for protein production, see,e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C.Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). Host cellsinclude cultured cells, e.g., mammalian cultured cells, yeast cells,insect cells, bacterial cells and plant cells, to name only a few, butalso cells comprised within a transgenic animal, transgenic plant orcultured plant or animal tissue. In one embodiment, the host cell is aeukaryotic cell, preferably a mammalian cell, such as a Chinese HamsterOvary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell(e.g., YO, NS0, Sp20 cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antigen binding domain such as anantibody, may be engineered so as to also express the other of theantibody chains such that the expressed product is an antibody that hasboth a heavy and a light chain.

In one embodiment, a method of producing a protease-activatable T cellactivating bispecific molecule according to the invention is provided,wherein the method comprises culturing a host cell comprising apolynucleotide encoding the protease-activatable T cell activatingbispecific molecule, as provided herein, under conditions suitable forexpression of the protease-activatable T cell activating bispecificmolecule, and recovering the protease-activatable T cell activatingbispecific molecule from the host cell (or host cell culture medium).

The components of the protease-activatable T cell activating bispecificmolecule are genetically fused to each other. Protease-activatable Tcell activating bispecific molecules can be designed such that itscomponents are fused directly to each other or indirectly through alinker sequence. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. Examples of linker sequences between differentcomponents of protease-activatable T cell activating bispecificmolecules are found in the sequences provided herein. Additionalsequences may also be included to incorporate a cleavage site toseparate the individual components of the fusion if desired, for examplean endopeptidase recognition sequence.

In certain embodiments the one or more antigen binding moieties of theprotease-activatable T cell activating bispecific molecules comprise atleast an antibody variable region capable of binding an antigenicdeterminant. Variable regions can form part of and be derived fromnaturally or non-naturally occurring antibodies and fragments thereof.Methods to produce polyclonal antibodies and monoclonal antibodies arewell known in the art (see e.g., Harlow and Lane, “Antibodies, alaboratory manual”, Cold Spring Harbor Laboratory, 1988). Non-naturallyoccurring antibodies can be constructed using solid phase-peptidesynthesis, can be produced recombinantly (e.g., as described in U.S.Pat. No. 4,186,567) or can be obtained, for example, by screeningcombinatorial libraries comprising variable heavy chains and variablelight chains (see e.g., U.S. Pat. No. 5,969,108 to McCafferty).

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region can be used in the protease-activatable T cellactivating bispecific molecules of the invention. Non-limitingantibodies, antibody fragments, antigen binding domains or variableregions useful in the present invention can be of murine, primate, orhuman origin. If the protease-activatable T cell activating bispecificmolecule is intended for human use, a chimeric form of antibody may beused wherein the constant regions of the antibody are from a human. Ahumanized or fully human form of the antibody can also be prepared inaccordance with methods well known in the art (see e. g. U.S. Pat. No.5,565,332 to Winter). Humanization may be achieved by various methodsincluding, but not limited to (a) grafting the non-human (e.g., donorantibody) CDRs onto human (e.g., recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g., those that are important for retaining good antigenbinding affinity or antibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (c) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332, 323-329(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones etal., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81,6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988);Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498(1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36, 43-60(2005) (describing “FR shuffling”); and Osbourn et al., Methods 36,61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000)(describing the “guided selection” approach to FR shuffling). Humanantibodies and human variable regions can be produced using varioustechniques known in the art. Human antibodies are described generally invan Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) andLonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regionscan form part of and be derived from human monoclonal antibodies made bythe hybridoma method (see e.g., Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987)). Human antibodies and human variable regions may also be preparedby administering an immunogen to a transgenic animal that has beenmodified to produce intact human antibodies or intact antibodies withhuman variable regions in response to antigenic challenge (see e.g.,Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies and humanvariable regions may also be generated by isolating Fv clone variableregion sequences selected from human-derived phage display libraries(see e.g., Hoogenboom et al. in Methods in Molecular Biology 178, 1-37(O'Brien et al., ed., Human Press, Totowa, N.J., 2001); and McCaffertyet al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628(1991)). Phage typically display antibody fragments, either assingle-chain Fv (scFv) fragments or as Fab fragments.

In certain embodiments, the antigen binding moieties useful in thepresent invention are engineered to have enhanced binding affinityaccording to, for example, the methods disclosed in U.S. Pat. Appl.Publ. No. 2004/0132066, the entire contents of which are herebyincorporated by reference. The ability of the protease-activatable Tcell activating bispecific molecule of the invention to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g., surface plasmon resonance technique(analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17,323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)). Competition assays may be used to identify an antibody,antibody fragment, antigen binding domain or variable domain thatcompetes with a reference antibody for binding to a particular antigen,e.g., an antibody that competes with the V9 antibody for binding to CD3.In certain embodiments, such a competing antibody binds to the sameepitope (e.g., a linear or a conformational epitope) that is bound bythe reference antibody. Detailed exemplary methods for mapping anepitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.). In an exemplary competition assay,immobilized antigen (e.g., CD3) is incubated in a solution comprising afirst labeled antibody that binds to the antigen (e.g., V9 antibody,described in U.S. Pat. No. 6,054,297) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to the antigen. The second antibody may be present in ahybridoma supernatant. As a control, immobilized antigen is incubated ina solution comprising the first labeled antibody but not the secondunlabeled antibody. After incubation under conditions permissive forbinding of the first antibody to the antigen, excess unbound antibody isremoved, and the amount of label associated with immobilized antigen ismeasured. If the amount of label associated with immobilized antigen issubstantially reduced in the test sample relative to the control sample,then that indicates that the second antibody is competing with the firstantibody for binding to the antigen. See Harlow and Lane (1988)Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.).

Protease-activatable T cell activating bispecific molecules prepared asdescribed herein may be purified by art-known techniques such as highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, affinity chromatography, size exclusion chromatography,and the like. The actual conditions used to purify a particular proteinwill depend, in part, on factors such as net charge, hydrophobicity,hydrophilicity etc., and will be apparent to those having skill in theart. For affinity chromatography purification an antibody, ligand,receptor or antigen can be used to which the protease-activatable T cellactivating bispecific molecule binds. For example, for affinitychromatography purification of protease-activatable T cell activatingbispecific molecules of the invention, a matrix with protein A orprotein G may be used. Sequential Protein A or G affinity chromatographyand size exclusion chromatography can be used to isolate aprotease-activatable T cell activating bispecific molecule essentiallyas described in the Examples. The purity of the protease-activatable Tcell activating bispecific molecule can be determined by any of avariety of well-known analytical methods including gel electrophoresis,high pressure liquid chromatography, and the like. For example, theheavy chain fusion proteins expressed as described in the Examples wereshown to be intact and properly assembled as demonstrated by reducingSDS-PAGE (see, e.g., FIGS. 8-12 ). Three bands were resolved atapproximately Mr 25,000, Mr 50,000 and Mr 75,000, corresponding to thepredicted molecular weights of the protease-activatable T cellactivating bispecific molecule light chain, heavy chain and heavychain/light chain fusion protein.

Assays

protease-activatable T cell activating bispecific molecules providedherein may be identified, screened for, or characterized for theirphysical/chemical properties and/or biological activities by variousassays known in the art.

Affinity Assays

The affinity of the protease-activatable T cell activating bispecificmolecule for an Fc receptor or a target antigen can be determined inaccordance with the methods set forth in the Examples by surface plasmonresonance (SPR), using standard instrumentation such as a BIAcoreinstrument (GE Healthcare), and receptors or target proteins such as maybe obtained by recombinant expression. Alternatively, binding ofprotease-activatable T cell activating bispecific molecules fordifferent receptors or target antigens may be evaluated using cell linesexpressing the particular receptor or target antigen, for example byflow cytometry (FACS). A specific illustrative and exemplary embodimentfor measuring binding affinity is described in the following and in theExamples below. According to one embodiment, K_(D) is measured bysurface plasmon resonance using a BIACORE® T100 machine (GE Healthcare)at 25° C.

To analyze the interaction between the Fc-portion and Fc receptors,His-tagged recombinant Fc-receptor is captured by an anti-Penta Hisantibody (Qiagen) immobilized on CMS chips and the bispecific constructsare used as analytes. Briefly, carboxymethylated dextran biosensor chips(CMS, GE Healthcare) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti Penta-His antibody is diluted with 10 mM sodium acetate, pH 5.0, to40 m/ml before injection at a flow rate of 5 μl/min to achieveapproximately 6500 response units (RU) of coupled protein. Following theinjection of the ligand, 1 M ethanolamine is injected to block unreactedgroups. Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM.For kinetic measurements, four-fold serial dilutions of the bispecificconstruct (range between 500 nM and 4000 nM) are injected in HBS-EP (GEHealthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20,pH 7.4) at 25° C. at a flow rate of 30 μl/min for 120 s.

To determine the affinity to the target antigen, bispecific constructsare captured by an anti-human Fab specific antibody (GE Healthcare) thatis immobilized on an activated CMS-sensor chip surface as described forthe anti Penta-His antibody. The final amount of coupled protein is isapproximately 12000 R U. The bispecific constructs are captured for 90 sat 300 nM. The target antigens are passed through the flow cells for 180s at a concentration range from 250 to 1000 nM with a flowrate of 30μl/min. The dissociation is monitored for 180 s.

Bulk refractive index differences are corrected for by subtracting theresponse obtained on reference flow cell. The steady state response wasused to derive the dissociation constant K_(D) by non-linear curvefitting of the Langmuir binding isotherm. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1)by simultaneously fitting the association and dissociation sensorgrams.The equilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

Activity Assays

Biological activity of the protease-activatable T cell activatingbispecific molecules of the invention can be measured by various assaysas described in the Examples. Biological activities may for exampleinclude the induction of proliferation of T cells, the induction ofsignaling in T cells, the induction of expression of activation markersin T cells, the induction of cytokine secretion by T cells, theinduction of lysis of target cells such as tumor cells, and theinduction of tumor regression and/or the improvement of survival.

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the protease-activatable T cell activating bispecificmolecules provided herein, e.g., for use in any of the below therapeuticmethods. In one embodiment, a pharmaceutical composition comprises anyof the protease-activatable T cell activating bispecific moleculesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical composition comprises any of theprotease-activatable T cell activating bispecific molecules providedherein and at least one additional therapeutic agent, e.g., as describedbelow.

Further provided is a method of producing a protease-activatable T cellactivating bispecific molecule of the invention in a form suitable foradministration in vivo, the method comprising (a) obtaining aprotease-activatable T cell activating bispecific molecule according tothe invention, and (b) formulating the protease-activatable T cellactivating bispecific molecule with at least one pharmaceuticallyacceptable carrier, whereby a preparation of protease-activatable T cellactivating bispecific molecule is formulated for administration in vivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more protease-activatable Tcell activating bispecific molecule dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that are generally non-toxic to recipients at the dosagesand concentrations employed, i.e. do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains at least one protease-activatable T cellactivating bispecific molecule and optionally an additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards or corresponding authorities in other countries.Preferred compositions are lyophilized formulations or aqueoussolutions. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, buffers, dispersion media, coatings,surfactants, antioxidants, preservatives (e.g., antibacterial agents,antifungal agents), isotonic agents, absorption delaying agents, salts,preservatives, antioxidants, proteins, drugs, drug stabilizers,polymers, gels, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). Except insofar as any conventional carrier is incompatiblewith the active ingredient, its use in the therapeutic or pharmaceuticalcompositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. Protease-activatable T cell activating bispecific moleculesof the present invention (and any additional therapeutic agent) can beadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrasplenically, intrarenally, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, intratumorally, intramuscularly, intraperitoneally,subcutaneously, subconjunctivally, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularally, orally,topically, locally, by inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g., liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference). Parenteral administration, inparticular intravenous injection, is most commonly used foradministering polypeptide molecules such as the protease-activatable Tcell activating bispecific molecules of the invention.

Parenteral compositions include those designed for administration byinjection, e.g., subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the protease-activatable T cell activating bispecificmolecules of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the protease-activatable T cellactivating bispecific molecules may be in powder form for constitutionwith a suitable vehicle, e.g., sterile pyrogen-free water, before use.Sterile injectable solutions are prepared by incorporating theprotease-activatable T cell activating bispecific molecules of theinvention in the required amount in the appropriate solvent with variousof the other ingredients enumerated below, as required. Sterility may bereadily accomplished, e.g., by filtration through sterile filtrationmembranes. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and/or the other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, suspensions or emulsion, the preferred methods of preparationare vacuum-drying or freeze-drying techniques which yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles,e.g., films, or microcapsules. In particular embodiments, prolongedabsorption of an injectable composition can be brought about by the usein the compositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, theprotease-activatable T cell activating bispecific molecules may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theprotease-activatable T cell activating bispecific molecules may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the protease-activatable T cellactivating bispecific molecules of the invention may be manufactured bymeans of conventional mixing, dissolving, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The protease-activatable T cell activating bispecific molecules may beformulated into a composition in a free acid or base, neutral or saltform. Pharmaceutically acceptable salts are salts that substantiallyretain the biological activity of the free acid or base. These includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Pharmaceutical salts tend to be more soluble inaqueous and other protic solvents than are the corresponding free baseforms.

Therapeutic Methods and Compositions

Any of the protease-activatable T cell activating bispecific moleculesprovided herein may be used in therapeutic methods. Protease-activatableT cell activating bispecific molecules of the invention can be used asimmunotherapeutic agents, for example in the treatment of cancers.

For use in therapeutic methods, protease-activatable T cell activatingbispecific molecules of the invention would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, protease-activatable T cell activating bispecificmolecules of the invention for use as a medicament are provided. Infurther aspects, protease-activatable T cell activating bispecificmolecules of the invention for use in treating a disease are provided.In certain embodiments, protease-activatable T cell activatingbispecific molecules of the invention for use in a method of treatmentare provided. In one embodiment, the invention provides aprotease-activatable T cell activating bispecific molecule as describedherein for use in the treatment of a disease in an individual in needthereof. In certain embodiments, the invention provides aprotease-activatable T cell activating bispecific molecule for use in amethod of treating an individual having a disease comprisingadministering to the individual a therapeutically effective amount ofthe protease-activatable T cell activating bispecific molecule. Incertain embodiments the disease to be treated is a proliferativedisorder. In a particular embodiment the disease is cancer. In certainembodiments the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In further embodiments, the invention provides aprotease-activatable T cell activating bispecific molecule as describedherein for use in inducing lysis of a target cell, particularly a tumorcell. In certain embodiments, the invention provides aprotease-activatable T cell activating bispecific molecule for use in amethod of inducing lysis of a target cell, particularly a tumor cell, inan individual comprising administering to the individual an effectiveamount of the protease-activatable T cell activating bispecific moleculeto induce lysis of a target cell. An “individual” according to any ofthe above embodiments is a mammal, preferably a human.

In a further aspect, the invention provides for the use of aprotease-activatable T cell activating bispecific molecule of theinvention in the manufacture or preparation of a medicament. In oneembodiment the medicament is for the treatment of a disease in anindividual in need thereof. In a further embodiment, the medicament isfor use in a method of treating a disease comprising administering to anindividual having the disease a therapeutically effective amount of themedicament. In certain embodiments the disease to be treated is aproliferative disorder. In a particular embodiment the disease iscancer. In one embodiment, the method further comprises administering tothe individual a therapeutically effective amount of at least oneadditional therapeutic agent, e.g., an anti-cancer agent if the diseaseto be treated is cancer. In a further embodiment, the medicament is forinducing lysis of a target cell, particularly a tumor cell. In still afurther embodiment, the medicament is for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of themedicament to induce lysis of a target cell. An “individual” accordingto any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease. In one embodiment, the method comprises administering to anindividual having such disease a therapeutically effective amount of aprotease-activatable T cell activating bispecific molecule of theinvention. In one embodiment a composition is administered to saidindividual, comprising the protease-activatable T cell activatingbispecific molecule of the invention in a pharmaceutically acceptableform. In certain embodiments the disease to be treated is aproliferative disorder. In a particular embodiment the disease iscancer. In certain embodiments the method further comprisesadministering to the individual a therapeutically effective amount of atleast one additional therapeutic agent, e.g., an anti-cancer agent ifthe disease to be treated is cancer. An “individual” according to any ofthe above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for inducing lysisof a target cell, particularly a tumor cell. In one embodiment themethod comprises contacting a target cell with a protease-activatable Tcell activating bispecific molecule of the invention in the presence ofa T cell, particularly a cytotoxic T cell. In a further aspect, a methodfor inducing lysis of a target cell, particularly a tumor cell, in anindividual is provided. In one such embodiment, the method comprisesadministering to the individual an effective amount of aprotease-activatable T cell activating bispecific molecule to inducelysis of a target cell. In one embodiment, an “individual” is a human.

In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that can be treated using aprotease-activatable T cell activating bispecific molecule of thepresent invention include, but are not limited to neoplasms located inthe: abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous system(central and peripheral), lymphatic system, pelvic, skin, soft tissue,spleen, thoracic region, and urogenital system. Also included arepre-cancerous conditions or lesions and cancer metastases. In certainembodiments the cancer is chosen from the group consisting of renal cellcancer, skin cancer, lung cancer, colorectal cancer, breast cancer,brain cancer, head and neck cancer. A skilled artisan readily recognizesthat in many cases the protease-activatable T cell activating bispecificmolecule may not provide a cure but may only provide partial benefit. Insome embodiments, a physiological change having some benefit is alsoconsidered therapeutically beneficial. Thus, in some embodiments, anamount of protease-activatable T cell activating bispecific moleculethat provides a physiological change is considered an “effective amount”or a “therapeutically effective amount”. The subject, patient, orindividual in need of treatment is typically a mammal, more specificallya human.

In some embodiments, an effective amount of a protease-activatable Tcell activating bispecific molecule of the invention is administered toa cell. In other embodiments, a therapeutically effective amount of aprotease-activatable T cell activating bispecific molecule of theinvention is administered to an individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of aprotease-activatable T cell activating bispecific molecule of theinvention (when used alone or in combination with one or more otheradditional therapeutic agents) will depend on the type of disease to betreated, the route of administration, the body weight of the patient,the type of T cell activating bispecific antigen binding molecule, theseverity and course of the disease, whether the T cell activatingbispecific antigen binding molecule is administered for preventive ortherapeutic purposes, previous or concurrent therapeutic interventions,the patient's clinical history and response to the protease-activatableT cell activating bispecific molecule, and the discretion of theattending physician. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.Various dosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

The protease-activatable T cell activating bispecific molecule issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of protease-activatable Tcell activating bispecific molecule can be an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the T cell activating bispecific antigenbinding molecule would be in the range from about 0.005 mg/kg to about10 mg/kg. In other non-limiting examples, a dose may also comprise fromabout 1 microgram/kg body weight, about 5 microgram/kg body weight,about 10 microgram/kg body weight, about 50 microgram/kg body weight,about 100 microgram/kg body weight, about 200 microgram/kg body weight,about 350 microgram/kg body weight, about 500 microgram/kg body weight,about 1 milligram/kg body weight, about 5 milligram/kg body weight,about 10 milligram/kg body weight, about 50 milligram/kg body weight,about 100 milligram/kg body weight, about 200 milligram/kg body weight,about 350 milligram/kg body weight, about 500 milligram/kg body weight,to about 1000 mg/kg body weight or more per administration, and anyrange derivable therein. In non-limiting examples of a derivable rangefrom the numbers listed herein, a range of about 5 mg/kg body weight toabout 100 mg/kg body weight, about 5 microgram/kg body weight to about500 milligram/kg body weight, etc., can be administered, based on thenumbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g., every week or every three weeks (e.g., such thatthe patient receives from about two to about twenty, or e.g., about sixdoses of the protease-activatable T cell activating bispecificmolecule). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

The protease-activatable T cell activating bispecific molecule of theinvention will generally be used in an amount effective to achieve theintended purpose. For use to treat or prevent a disease condition, theprotease-activatable T cell activating bispecific molecules of theinvention, or pharmaceutical compositions thereof, are administered orapplied in a therapeutically effective amount. Determination of atherapeutically effective amount is well within the capabilities ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the protease-activatable T cell activating bispecificmolecules which are sufficient to maintain therapeutic effect. Usualpatient dosages for administration by injection range from about 0.1 to50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC. Incases of local administration or selective uptake, the effective localconcentration of the protease-activatable T cell activating bispecificmolecules may not be related to plasma concentration. One having skillin the art will be able to optimize therapeutically effective localdosages without undue experimentation.

A therapeutically effective dose of the protease-activatable T cellactivating bispecific molecules described herein will generally providetherapeutic benefit without causing substantial toxicity. Toxicity andtherapeutic efficacy of a protease-activatable T cell activatingbispecific molecule can be determined by standard pharmaceuticalprocedures in cell culture or experimental animals. Cell culture assaysand animal studies can be used to determine the LD₅₀ (the dose lethal to50% of a population) and the ED₅₀ (the dose therapeutically effective in50% of a population). The dose ratio between toxic and therapeuticeffects is the therapeutic index, which can be expressed as the ratioLD₅₀/ED₅₀. Protease-activatable T cell activating bispecific moleculethat exhibit large therapeutic indices are preferred. In one embodiment,the protease-activatable T cell activating bispecific molecule accordingto the present invention exhibits a high therapeutic index. The dataobtained from cell culture assays and animal studies can be used informulating a range of dosages suitable for use in humans. The dosagelies preferably within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon a variety of factors, e.g., the dosage formemployed, the route of administration utilized, the condition of thesubject, and the like. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition (see, e.g., Fingl et al., 1975, in: ThePharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated hereinby reference in its entirety). The attending physician for patientstreated with protease-activatable T cell activating bispecific moleculesof the invention would know how and when to terminate, interrupt, oradjust administration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Other Agents and Treatments

The protease-activatable T cell activating bispecific molecules of theinvention may be administered in combination with one or more otheragents in therapy. For instance, a protease-activatable T cellactivating bispecific molecule of the invention may be co-administeredwith at least one additional therapeutic agent. The term “therapeuticagent” encompasses any agent administered to treat a symptom or diseasein an individual in need of such treatment. Such additional therapeuticagent may comprise any active ingredients suitable for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. In certain embodiments, anadditional therapeutic agent is an immunomodulatory agent, a cytostaticagent, an inhibitor of cell adhesion, a cytotoxic agent, an activator ofcell apoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of protease-activatable T cellactivating bispecific molecule used, the type of disorder or treatment,and other factors discussed above. The protease-activatable T cellactivating bispecific molecule are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the protease-activatable T cell activating bispecificmolecule of the invention can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent and/oradjuvant. Protease-activatable T cell activating bispecific molecules ofthe invention can also be used in combination with radiation therapy.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a protease-activatable T cell activating bispecificmolecule of the invention. The label or package insert indicates thatthe composition is used for treating the condition of choice. Moreover,the article of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises aprotease-activatable T cell activating bispecific molecule of theinvention; and (b) a second container with a composition containedtherein, wherein the composition comprises a further cytotoxic orotherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

EXEMPLARY EMBODIMENTS

-   -   1. A protease-activatable T cell activating bispecific molecule        comprising        -   (a) a first antigen binding moiety capable of specific            binding to CD3;        -   (b) a second antigen binding moiety capable of specific            binding to a target cell antigen; and        -   (c) a masking moiety covalently attached to the T cell            bispecific binding molecule through a protease-cleavable            linker, wherein the masking moiety is capable of specific            binding to the idiotype of the first or the second antigen            binding moiety thereby reversibly concealing the first or            the second antigen binding moiety.    -   2. The protease-activatable T cell activating bispecific        molecule of embodiment 1, wherein the masking moiety is        covalently attached to the first antigen binding moiety and        reversibly conceals the first antigen binding moiety.    -   3. The protease-activatable T cell activating bispecific        molecule of embodiment 1 or 2, wherein the masking moiety is        covalently attached to the heavy chain variable region of the        first antigen binding moiety.    -   4. The protease-activatable T cell activating bispecific        molecule of embodiment 1 or 2, wherein the masking moiety is        covalently attached to the light chain variable region of the        first antigen binding moiety.    -   5. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 4, wherein the masking        moiety is an anti-idiotypic scFv.    -   6. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 2 to 5, wherein the        protease-activatable T cell activating bispecific molecule        comprises a second masking moiety reversibly concealing the        second antigen binding moiety.    -   7. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 6, wherein the protease        is expressed by the target cell.    -   8. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 7, wherein the second        antigen binding moiety is a crossover Fab molecule wherein        either the variable or the constant regions of the Fab light        chain and the Fab heavy chain are exchanged.    -   9. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 8, wherein the second        antigen binding moiety is a crossover Fab molecule wherein the        constant regions of the Fab light chain and the Fab heavy chain        are exchanged.    -   10. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 9, wherein the first        antigen binding moiety is a conventional Fab molecule.    -   11. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 10, comprising not more        than one antigen binding moiety capable of specific binding to        CD3.    -   12. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 11, comprising a third        antigen binding moiety which is a Fab molecule capable of        specific binding to a target cell antigen.    -   13. The protease-activatable T cell activating bispecific        molecule of embodiment 12, wherein the third antigen binding        moiety is identical to the second antigen binding moiety.    -   14. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 13, wherein the second        antigen binding moiety is capable of specific binding to FolR1        or HER.    -   15. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 13, wherein the second        antigen binding moiety is capable of specific binding to a        target cell antigen selected from the group consisting of FolR1,        HER1 and Mesothelin.    -   16. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 13, wherein the second        antigen binding moiety is capable of specific binding to a        target cell antigen selected from the group consisting of FolR1,        HER1, HER2 and Mesothelin.    -   17. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 16, wherein the first        and the second antigen binding moiety are fused to each other,        optionally via a peptide linker.    -   18. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 17, wherein the second        antigen binding moiety is fused at the C-terminus of the Fab        heavy chain to the N-terminus of the Fab heavy chain of the        first antigen binding moiety.    -   19. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 17, wherein the first        antigen binding moiety is fused at the C-terminus of the Fab        heavy chain to the N-terminus of the Fab heavy chain of the        second antigen binding moiety.    -   20. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 19, wherein the Fab        light chain of the first antigen binding moiety and the Fab        light chain of the second antigen binding moiety are fused to        each other, optionally via a peptide linker.    -   21. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 20, additionally        comprising an Fc domain composed of a first and a second subunit        capable of stable association.    -   22. The protease-activatable T cell activating bispecific        molecule of embodiment 21, wherein the Fc domain is an IgG,        specifically an IgG₁ or IgG4, Fc domain.    -   23. The protease-activatable T cell activating bispecific        molecule of embodiment 21 or 22, wherein the Fc domain is a        human Fc domain.    -   24. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 21 to 23, wherein the Fc        domain exhibits reduced binding affinity to an Fc receptor        and/or reduced effector function, as compared to a native IgG₁        Fc domain.    -   25. The protease-activatable T cell activating bispecific        molecule of embodiment 24, wherein the Fc domain comprises one        or more amino acid substitution that reduces binding to an Fc        receptor and/or effector function.    -   26. The protease-activatable T cell activating bispecific        molecule of embodiment 25, wherein said one or more amino acid        substitution is at one or more position selected from the group        of L234, L235, and P329 (Kabat numbering).    -   27. The protease-activatable T cell activating bispecific        molecule of embodiment 26, wherein each subunit of the Fc domain        comprises three amino acid substitutions that reduce binding to        an activating Fc receptor and/or effector function wherein said        amino acid substitutions are L234A, L235A and P329G.    -   28. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 24 to 27, wherein the Fc        receptor is an Fcγ receptor.    -   29. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 24 to 28, wherein the        effector function is antibody-dependent cell-mediated        cytotoxicity (ADCC).    -   30. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 29, wherein the masking        moiety comprises a heavy chain variable region comprising at        least one of:        -   (a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of DYSIH (SEQ ID NO:20);        -   (b) a CDR H2 amino acid sequence of WINTETGEPAYADDFKG (SEQ            ID NO:21); and        -   (c) a CDR H3 amino acid sequence of PYDYDVLDY (SEQ ID            NO:22).    -   31. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 30, wherein the masking        moiety comprises a light chain variable region comprising at        least one of:        -   (d) a light chain (CDR L)1 amino acid sequence of            RASKSVSTSNYSYIH (SEQ ID NO:23);        -   (e) a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24);            and        -   (f) a CDR L3 amino acid sequence of QHSREFPWT (SEQ ID            NO:25).    -   32. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 31, wherein the masking        moiety comprises a heavy chain variable region comprising:        -   (a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of DYSIH (SEQ ID NO:20);        -   (b) a CDR H2 amino acid sequence of WINTETGEPAYADDFKG (SEQ            ID NO:21);        -   (c) a CDR H3 amino acid sequence of PYDYDVLDY (SEQ ID            NO:22); and a light chain variable region comprising:        -   (d) a light chain (CDR L)1 amino acid sequence of            RASKSVSTSNYSYIH (SEQ ID NO:23);        -   (e) a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24);            and        -   (f) a CDR L3 amino acid sequence of QHSREFPWT (SEQ ID            NO:25).    -   33. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 29, wherein the masking        moiety comprises a heavy chain variable region comprising at        least one of:        -   (a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of SYGVS (SEQ ID NO:26);        -   (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID            NO:27); and        -   (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID            NO:28).    -   34. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 29 and 33, wherein the        masking moiety comprises a light chain variable region        comprising at least one of:        -   (d) a light chain (CDR L)1 amino acid sequence of            RASENIDSYLA (SEQ ID NO:29);        -   (e) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30);            and        -   (f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID            NO:31).    -   35. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 29 and 33 to 34, wherein        the masking moiety comprises a heavy chain variable region        comprising:        -   (a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of SYGVS (SEQ ID NO:26);        -   (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID            NO:27);        -   (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID            NO:28); and a light chain variable region comprising:        -   (d) a light chain (CDR L)1 amino acid sequence of            RASENIDSYLA (SEQ ID NO:29);        -   (e) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30);            and        -   (f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID            NO:31).    -   36. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 35, wherein the masking        moiety comprises a heavy chain variable region comprising at        least one of:        -   (a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of DYYIN (SEQ ID NO:48);        -   (b) a CDR H2 amino acid sequence of VINPDSGGTDYNQNFKG (SEQ            ID NO:49); and        -   (c) a CDR H3 amino acid sequence of RDSYGFDY (SEQ ID NO:50).    -   37. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 36, wherein the masking        moiety comprises a light chain variable region comprising at        least one of:        -   (a) a light chain (CDR L)1 amino acid sequence of            KASLSVTNDVA (SEQ ID NO:51);        -   (b) a CDR L2 amino acid sequence of YASNRNA (SEQ ID NO:52);            and        -   (c) a CDR L3 amino acid sequence of QQDYTSPPT (SEQ ID            NO:53).    -   38. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 37, wherein the masking        moiety comprises a heavy chain variable region comprising:        -   a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of DYYIN (SEQ ID NO:48);        -   b) a CDR H2 amino acid sequence of VINPDSGGTDYNQNFKG (SEQ ID            NO:49); and        -   c) a CDR H3 amino acid sequence of RDSYGFDY (SEQ ID NO:50);            and a light chain variable region comprising:        -   d) a light chain (CDR L)1 amino acid sequence of KASLSVTNDVA            (SEQ ID NO:51);        -   e) a CDR L2 amino acid sequence of YASNRNA (SEQ ID NO:52);            and        -   f) a CDR L3 amino acid sequence of QQDYTSPPT (SEQ ID NO:53).    -   39. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 38, wherein the protease        cleavable linker comprises at least one protease recognition        sequence.    -   40. The protease-activatable T cell activating bispecific        molecule of embodiment 39, wherein the protease cleavable linker        comprises a protease recognition sequence.    -   41. The protease-activatable T cell activating bispecific        molecule of embodiment 40, wherein the protease recognition        sequence is selected from the group consisting of:        -   (a) RQARVVNG (SEQ ID NO:36);        -   (b) VHMPLGFLGPGRSRGSFP (SEQ ID NO:37);        -   (c) RQARVVNGXXXXXVPLSLYSG (SEQ ID NO:38); and        -   (d) RQARVVNGVPLSLYSG (SEQ ID NO:39)        -   (e) PLGLWSQ (SEQ ID NO:40), wherein X is any amino acid.    -   42. The protease-activatable T cell activating bispecific        molecule of embodiment 40, wherein the protease recognition        sequence is selected from the group consisting of:        -   (a) RQARVVNG (SEQ ID NO:36);        -   (b) VHMPLGFLGPGRSRGSFP (SEQ ID NO:37);        -   (c) RQARVVNGXXXXXVPLSLYSG (SEQ ID NO:38);        -   (d) RQARVVNGVPLSLYSG (SEQ ID NO:39);        -   (e) PLGLWSQ (SEQ ID NO:40);        -   (f) VHMPLGFLGPRQARVVNG (SEQ ID NO:97);        -   (g) FVGGTG (SEQ ID NO:98);        -   (h) KKAAPVNG (SEQ ID NO:99);        -   (i) PMAKKVNG (SEQ ID NO:100);        -   (j) QARAKVNG (SEQ ID NO:101);        -   (k) VHMPLGFLGP (SEQ ID NO:102);        -   (l) QARAK (SEQ ID NO:103);        -   (m) VHMPLGFLGPPMAKK (SEQ ID NO:104);        -   (n) KKAAP (SEQ ID NO:105); and        -   (o) PMAKK (SEQ ID NO:106), wherein X is any amino acid.    -   43. The protease-activatable T cell activating bispecific        molecule of embodiment 39 or 40, wherein the protease cleavable        linker comprises the protease recognition sequence RQARVVNG (SEQ        ID NO:36).    -   44. The protease-activatable T cell activating bispecific        molecule of embodiment 39 or 40, wherein the protease cleavable        linker comprises the protease recognition sequence        VHMPLGFLGPRQARVVNG (SEQ ID NO:97).    -   45. The protease-activatable T cell activating bispecific        molecule of embodiment 39 or 40, wherein the protease cleavable        linker comprises the protease recognition sequence RQARVVNG (SEQ        ID NO:36) or the protease recognition sequence        VHMPLGFLGPRQARVVNG (SEQ ID NO:97).    -   46. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 45, wherein the protease        is selected from the group consisting of metalloproteinase,        serine protease, cysteine protease, aspartic proteases, and        cathepsin protease.    -   47. The protease-activatable T cell activating bispecific        molecule of embodiment 46, wherein the metalloproteinase is a        matrix metalloproteinase (MMP), preferably MMP9 or MMP2.    -   48. The protease-activatable T cell activating bispecific        molecule of embodiment 46, wherein the serine protease is        Matriptase.    -   49. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, wherein the first        antigen binding moiety comprises at least one heavy chain        complementarity determining region (CDR) selected from the group        consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and        at least one light chain CDR selected from the group of SEQ ID        NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.    -   50. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 49, wherein the first        antigen binding moiety is capable of specific binding to CD3 and        comprises a heavy chain variable region comprising:        -   a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of TYAMN (SEQ ID NO:44);        -   b) a CDR H2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ            ID NO:45); and        -   c) a CDR H3 amino acid sequence of HGNFGNSYVSWFAY (SEQ ID            NO:46); and a light chain variable region comprising:        -   d) a light chain (CDR L)1 amino acid sequence of            GSSTGAVTTSNYAN (SEQ ID NO:17);        -   e) a CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO:18);            and        -   f) a CDR L3 amino acid sequence of ALWYSNLWV (SEQ ID NO:19).    -   51. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 50, wherein the first        antigen binding moiety comprises a heavy chain variable region        comprising an amino acid sequence that is at least about 95%,        96%, 97%, 98%, 99% or 100% identical to the amino acid sequence        of SEQ ID NO: 43 and a light chain variable region comprising an        amino acid sequence that is at least about 95%, 96%, 97%, 98%,        99% or 100% identical to the amino acid sequence of SEQ ID NO:        55.    -   52. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 50, wherein the first        antigen binding moiety is capable of specific binding to CD3 and        comprises a heavy chain variable region comprising the amino        acid sequence of SEQ ID NO: 43 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 55.    -   53. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52, wherein the second        antigen binding moiety is capable of specific binding to FolR1        and comprises at least one heavy chain complementarity        determining region (CDR) selected from the group consisting of        SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at least one        light chain CDR selected from the group of SEQ ID NO: 17, SEQ ID        NO: 18 and SEQ ID NO: 19.    -   54. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 53, wherein the second        antigen binding moiety is capable of specific binding to FolR1        and comprises a heavy chain variable region comprising:        -   a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of NAWMS (SEQ ID NO:14);        -   b) a CDR H2 amino acid sequence of RIKSKTDGGTTDYAAPVKG (SEQ            ID NO:15); and        -   c) a CDR H3 amino acid sequence of PWEWSWYDY (SEQ ID NO:16);            and a light chain variable region comprising:        -   d) a light chain (CDR L)1 amino acid sequence of            GSSTGAVTTSNYAN (SEQ ID NO:17);        -   e) a CDR L2 amino acid sequence of GTNKRAP (SEQ ID NO:18);            and        -   f) a CDR L3 amino acid sequence of ALWYSNLWV (SEQ ID NO:19).    -   55. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 54, wherein the second        antigen binding moiety comprises a heavy chain variable region        comprising an amino acid sequence that is at least about 95%,        96%, 97%, 98%, 99% or 100% identical to the amino acid sequence        of SEQ ID NO: 47 and a light chain variable region comprising an        amino acid sequence that is at least about 95%, 96%, 97%, 98%,        99% or 100% identical to the amino acid sequence of SEQ ID NO:        55.    -   56. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 54, wherein the second        antigen binding moiety is capable of specific binding to FolR1        and comprises a heavy chain variable region comprising the amino        acid sequence of SEQ ID NO: 47 and a light chain variable region        comprising the amino acid sequence of SEQ ID NO: 55.    -   57. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52, wherein the second        antigen binding moiety is capable of specific binding to FolR1        and comprises at least one heavy chain complementarity        determining region (CDR) selected from the group consisting of        SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153 and at least        one light chain CDR selected from the group of SEQ ID NO: 154,        SEQ ID NO: 155 and SEQ ID NO: 156.    -   58. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52 or 57, wherein the        second antigen binding moiety is capable of specific binding to        FolR1 and comprises a heavy chain variable region comprising:        -   a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of SYYMH (SEQ ID NO:151);        -   b) a CDR H2 amino acid sequence of IINPSGGSTSYAQKFQG (SEQ ID            NO:152); and        -   c) a CDR H3 amino acid sequence of SFFTGFHLDY (SEQ ID            NO:153); and a light chain variable region comprising:        -   d) a light chain (CDR L)1 amino acid sequence of            RASQSVSSSYLA (SEQ ID NO:154);        -   e) a CDR L2 amino acid sequence of GASSRAT (SEQ ID NO:155);            and        -   f) a CDR L3 amino acid sequence of QQYTNEHYYT (SEQ ID            NO:156).    -   59. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52, 57 or 58, wherein        the second antigen binding moiety comprises a heavy chain        variable region comprising an amino acid sequence that is at        least about 95%, 96%, 97%, 98%, 99% or 100% identical to the        amino acid sequence of SEQ ID NO: 157 and a light chain variable        region comprising an amino acid sequence that is at least about        95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid        sequence of SEQ ID NO: 158.    -   60. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52 or 57 to 59, wherein        the second antigen binding moiety wherein the second antigen        binding moiety is capable of specific binding to ForR1 and        comprises a heavy chain variable region comprising the amino        acid sequence of SEQ ID NO: 157 and a light chain variable        region comprising the amino acid sequence of SEQ ID NO: 158.    -   61. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52, wherein the second        antigen binding moiety is capable of specific binding to        Mesothelin and comprises at least one heavy chain        complementarity determining region (CDR) selected from the group        consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109        and at least one light chain CDR selected from the group of SEQ        ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112.    -   62. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52 or 61, wherein the        second antigen binding moiety is capable of specific binding to        Mesothelin and comprises a heavy chain variable region        comprising:        -   a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of GYTMN (SEQ ID NO:107);        -   b) a CDR H2 amino acid sequence of LITPYNGASSYNQKFRG (SEQ ID            NO:108); and        -   c) a CDR H3 amino acid sequence of GGYDGRGFDY (SEQ ID            NO:109); and a light chain variable region comprising:        -   d) a light chain (CDR L)1 amino acid sequence of SASSSVSYMH            (SEQ ID NO:110);        -   e) a CDR L2 amino acid sequence of DTSKLAS (SEQ ID NO:111);            and        -   f) a CDR L3 amino acid sequence of QQWSKHPLT (SEQ ID            NO:112).    -   63. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52, 61 or 62, wherein        the second antigen binding moiety comprises a heavy chain        variable region comprising an amino acid sequence that is at        least about 95%, 96%, 97%, 98%, 99% or 100% identical to the        amino acid sequence of SEQ ID NO: 113 and a light chain variable        region comprising an amino acid sequence that is at least about        95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid        sequence of SEQ ID NO: 114.    -   64. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52 or 61 to 63, wherein        the second antigen binding moiety is capable of specific binding        to Mesothelin and comprises a heavy chain variable region        comprising the amino acid sequence of SEQ ID NO: 113 and a light        chain variable region comprising to the amino acid sequence of        SEQ ID NO: 114.    -   65. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52, wherein the second        antigen binding moiety comprises a heavy chain comprising an        amino acid sequence that is at least about 95%, 96%, 97%, 98%,        99% or 100% identical to the amino acid sequence of SEQ ID NO:        32 and a light chain comprising an amino acid sequence that is        at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the        amino acid sequence of SEQ ID NO: 33.    -   66. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 52 or 65, wherein the        second antigen binding moiety is capable of specific binding to        HER1 and comprises a heavy chain variable region comprising the        amino acid sequence of SEQ ID NO: 115 and a light chain variable        region comprising the amino acid sequence of SEQ ID NO: 116.    -   67. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   (a) a first heavy chain comprising the amino acid sequence            of SEQ ID NO:2;        -   (b) a second heavy chain comprising the amino acid sequence            of SEQ ID NO:3; and        -   (c) a light chain comprising an amino acid sequence of SEQ            ID NO:1.    -   68. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   (a) a first heavy chain comprising the amino acid sequence            of SEQ ID NO:4;        -   (b) a second heavy chain comprising the amino acid sequence            of SEQ ID NO:3; and        -   (c) a light chain comprising an amino acid sequence of SEQ            ID NO:1.    -   69. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   a) at least one heavy chain comprising the amino acid            sequence of SEQ ID NO:32;        -   b) at least one light chain comprising the amino acid            sequence of SEQ ID NO:34.    -   70. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   (a) a first heavy chain comprising the amino acid sequence            of SEQ ID NO:72;        -   (b) a second heavy chain comprising the amino acid sequence            of SEQ ID NO:3; and    -   (c) a light chain comprising an amino acid sequence of SEQ ID        NO:1.    -   71. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   (a) a first heavy chain comprising the amino acid sequence            of SEQ ID NO:85;        -   (b) a second heavy chain comprising the amino acid sequence            of SEQ ID NO:3; and        -   (c) a light chain comprising an amino acid sequence of SEQ            ID NO:1.    -   72. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   (a) a first heavy chain comprising the amino acid sequence            of SEQ ID NO:73;        -   (b) a second heavy chain comprising the amino acid sequence            of SEQ ID NO:3;        -   (c) a first light chain comprising an amino acid sequence of            SEQ ID NO:1; and        -   (d) a second light chain comprising an amino acid sequence            of SEQ ID NO: 74.    -   73. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   (a) a first heavy chain comprising the amino acid sequence            of SEQ ID NO:77;        -   (b) a second heavy chain comprising the amino acid sequence            of SEQ ID NO:82;        -   (c) a first light chain comprising an amino acid sequence of            SEQ ID NO:78; and        -   (d) a second light chain comprising an amino acid sequence            of SEQ ID NO:81.    -   74. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   (a) a first heavy chain comprising the amino acid sequence            of SEQ ID NO:76;        -   (b) a second heavy chain comprising the amino acid sequence            of SEQ ID NO:77;        -   (c) a first light chain comprising an amino acid sequence of            SEQ ID NO:78; and        -   (d) a second light chain comprising an amino acid sequence            of SEQ ID NO:79.    -   75. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   (a) a first heavy chain comprising the amino acid sequence            of SEQ ID NO:132;        -   (b) a second heavy chain comprising the amino acid sequence            of SEQ ID NO:136;        -   (c) a first light chain comprising an amino acid sequence of            SEQ ID NO:81; and        -   (d) a second light chain comprising an amino acid sequence            of SEQ ID NO:133.    -   76. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 48, comprising        -   (a) a first heavy chain comprising the amino acid sequence            of SEQ ID NO:137;        -   (b) a second heavy chain comprising the amino acid sequence            of SEQ ID NO:139;        -   (c) a first light chain comprising an amino acid sequence of            SEQ ID NO:81; and        -   (d) a second light chain comprising an amino acid sequence            of SEQ ID NO:138.    -   77. An idiotype-specific polypeptide for reversibly concealing        an anti-CD3 antigen binding site of a molecule.    -   78. The idiotype-specific polypeptide of embodiment 77, wherein        the idiotype-specific polypeptide is an anti-idiotype scFv.    -   79. The idiotype-specific polypeptide of embodiment 77 or 78,        wherein the idiotype-specific polypeptide is covalently attached        to the molecule through a linker.    -   80. The idiotype-specific polypeptide of embodiment 79, wherein        the linker is a peptide linker.    -   81. The idiotype-specific polypeptide of embodiment 79 or 80,        wherein the linker is a protease-cleavable linker.    -   82. The idiotype-specific polypeptide of any one of embodiments        79 to 81, wherein the peptide linker comprises at least one        protease recognition site.    -   83. The idiotype-specific polypeptide of embodiment 82, wherein        the protease is selected from the group consisting of        metalloproteinase, serine protease, cysteine protease, aspartic        proteases, and cathepsin protease.    -   84. The idiotype-specific polypeptide of embodiment 83, wherein        the metalloproteinase is a matrix metalloproteinase (MMP),        preferably MMP9 or MMP2.    -   85. The idiotype-specific polypeptide of embodiment 83, wherein        the serine protease is

Matriptase.

-   -   86. The idiotype-specific polypeptide of embodiment 82, wherein        the protease cleavable linker comprises the protease recognition        sequence RQARVVNG (SEQ ID NO:36) or the protease recognition        sequence VHMPLGFLGPRQARVVNG (SEQ ID NO:97).    -   87. The idiotype-specific polypeptide of any one of embodiments        77 to 86, wherein the molecule is a T-cell activating bispecific        molecule.    -   88. The idiotype-specific polypeptide of any one of embodiments        77 to 87, comprising a heavy chain variable region comprising at        least one of:        -   (a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of DYSIH (SEQ ID NO:20);        -   (b) a CDR H2 amino acid sequence of WINTETGEPAYADDFKG (SEQ            ID NO:21); and        -   (c) a CDR H3 amino acid sequence of PYDYDVLDY (SEQ ID            NO:22).    -   89. The idiotype-specific polypeptide of any one of embodiments        77 to 88, comprising a light chain variable region comprising at        least one of:        -   (d) a light chain (CDR L)1 amino acid sequence of            RASKSVSTSNYSYIH (SEQ ID NO:23);        -   (e) a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24);            and        -   (f) a CDR L3 amino acid sequence of QHSREFPWT (SEQ ID            NO:25).    -   90. The idiotype-specific polypeptide of any one of embodiments        77 to 87, comprising a heavy chain variable region comprising:        -   (a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of DYSIH (SEQ ID NO:20);        -   (b) a CDR H2 amino acid sequence of WINTETGEPAYADDFKG (SEQ            ID NO:21);        -   (c) a CDR H3 amino acid sequence of PYDYDVLDY (SEQ ID            NO:22); and a light chain variable region comprising:        -   (d) a light chain (CDR L)1 amino acid sequence of            RASKSVSTSNYSYIH (SEQ ID NO:23);        -   (e) a CDR L2 amino acid sequence of YVSYLES (SEQ ID NO:24);            and        -   (f) a CDR L3 amino acid sequence of QHSREFPWT (SEQ ID            NO:25).    -   91. The idiotype-specific polypeptide of any one of embodiments        77 to 87, comprising a heavy chain variable region comprising at        least one of:        -   (a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of SYGVS (SEQ ID NO:26);        -   (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID            NO:27); and        -   (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID            NO:28).    -   92. The idiotype-specific polypeptide of any one of embodiments        77 to 87 and 91, comprising a light chain variable region        comprising at least one of:        -   (d) a light chain (CDR L)1 amino acid sequence of            RASENIDSYLA (SEQ ID NO:29);        -   (e) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30);            and        -   (f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID            NO:31).    -   93. The idiotype-specific polypeptide of any one of embodiments        77 to 87, comprising a heavy chain variable region comprising:        -   (a) a heavy chain complementarity determining region (CDR H)            1 amino acid sequence of SYGVS (SEQ ID NO:26);        -   (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID            NO:27);        -   (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID            NO:28); and a light chain variable region comprising:        -   (d) a light chain (CDR L)1 amino acid sequence of            RASENIDSYLA (SEQ ID NO:29);        -   (e) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30);            and        -   (f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID            NO:31).    -   94. The idiotype-specific polypeptide of embodiments 77 to 93,        wherein the anti-CD3 antigen binding site comprises a heavy        chain variable region comprising the amino acid sequence of SEQ        ID NO: 43 and a light chain variable region comprising the amino        acid sequence of SEQ ID NO: 55.    -   95. An isolated polynucleotide encoding the protease-activatable        T cell activating bispecific antigen binding molecule of any one        of embodiments 1 to 76 or the idiotype-specific polypeptide of        any one of embodiments 77 to 94.    -   96. A polypeptide encoded by the polynucleotide of embodiment        95.    -   97. A vector, particularly an expression vector, comprising the        polynucleotide of embodiment 95.    -   98. A host cell comprising the polynucleotide of embodiment 95        or the vector of embodiment 97.    -   99. A method of producing a protease-activatable T cell        activating bispecific molecule, comprising the steps of a)        culturing the host cell of embodiment 98 under conditions        suitable for the expression of the protease-activatable T cell        activating bispecific molecule and b) recovering the        protease-activatable T cell activating bispecific molecule.    -   100. A protease-activatable T cell activating bispecific        molecule produced by the method of embodiment 99.    -   101. A method of producing an idiotype-specific polypeptide,        comprising the steps of a) culturing the host cell of embodiment        98 under conditions suitable for the expression of the        idiotype-specific polypeptide and b) recovering the an        idiotype-specific polypeptide.    -   102. An idiotype-specific polypeptide produced by the method of        embodiment 101.    -   103. A pharmaceutical composition comprising the        protease-activatable T cell activating bispecific molecule of        any one of embodiments 1 to 76 and a pharmaceutically acceptable        carrier.    -   104. A pharmaceutical composition comprising the        idiotype-specific polypeptide of any one of embodiments 77 to 94        and a pharmaceutically acceptable carrier.    -   105. A protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 76, the        idiotype-specific polypeptide of any one of embodiments 77 to 94        or the composition of embodiment 103 for use as a medicament.    -   106. The protease-activatable T cell activating bispecific        molecule for use according to embodiment 105, wherein the        medicament is for treating or delaying progression of cancer,        treating or delaying progression of an immune related disease,        or enhancing or stimulating an immune response or function in an        individual.    -   107. The protease-activatable T cell activating bispecific        molecule of any one of embodiments 1 to 76 or the        idiotype-specific polypeptide of any one of embodiments 77 to 94        for use in the treatment of a disease in an individual in need        thereof.    -   108. The protease-activatable T cell activating bispecific        molecule or the idiotype-specific polypeptide for use in the        treatment of a disease in an individual in need thereof of        embodiment 107, wherein the disease is a cancer.    -   109. Use of the protease-activatable T cell activating        bispecific molecule of any one of embodiments 1 to 76 or the        idiotype-specific polypeptide of any one of embodiments 77 to 94        for the manufacture of a medicament.    -   110. The use of embodiment 109, wherein the disease is a cancer.    -   111. A method of treating a disease in an individual, comprising        administering to said individual a therapeutically effective        amount of a composition comprising the protease-activatable T        cell activating bispecific molecule of any one of embodiments 1        to 76 or composition of embodiment 103.    -   112. A method for inducing lysis of a target cell, comprising        contacting a target cell with the protease-activatable T cell        activating bispecific molecule of any one of embodiments 1 to 76        or composition of embodiment 103 in the presence of a T cell.    -   113. The method of embodiment 112 wherein the target cell is a        cancer cell.    -   114. The method of embodiment 112 or 113, wherein the target        cell expresses a protease capable of activating the        protease-activatable T cell activating bispecific molecule.    -   115. An anti-idiotype CD3 antibody or antigen-binding fragment        thereof specific for an idiotype of an anti-CD3 antigen-binding        molecule, wherein the anti-idiotype CD3 antibody or fragment        thereof when bound to the anti-CD3 antigen-binding molecule        specifically blocks binding of the anti-CD3 antigen-binding        molecule to CD3.    -   116. The anti-idiotype CD3 antibody or antigen-binding fragment        thereof of embodiment 115, wherein the anti-idiotype CD3        antibody or fragment thereof is reversibly associated with the        anti-CD3 antigen-binding molecule through a peptide linker        comprising a protease recognition site.    -   117. The anti-idiotype CD3 antibody or antigen-binding fragment        thereof of embodiment 115 or 116, wherein the CD3 is a mouse,        monkey or human CD3.    -   118. A method of reducing in vivo toxicity of a T cell        activating bispecific molecule comprising attaching an        idiotype-specific polypeptide of any one of embodiments 77 to 94        to the T cell activating bispecific molecule with a        protease-cleavable linker to form a protease-activatable T cell        activating bispecific molecule, wherein the in vivo toxicity of        the protease-activatable T cell activating bispecific molecule        is reduced compared to toxicity of the T cell activating        bispecific molecule.    -   119. The invention as described hereinbefore.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Synthesis of Monovalent Anti-CD3 IgG Molecules withAnti-Idiotypic scFv

Described herein are CD3 binders that are masked with an N-terminallylinked anti-idiotypic CD3 scFv. These constructs include a proteaserecognition site which is recognized by a tumor specific protease. Inthe presence of protease-expressing tumor cells, the linker connectingthe masking moiety will be cleaved and, thereby, CD3 binding by the CD3binder is recovered. Several monovalent anti-CD3 IgG molecules withvarious anti-idiotypic scFv were produced and are schematically depictedin FIGS. 1A-1E with their respective ID number. The following moleculeswere prepared:

-   -   Identification No. 7859: monovalent CD3 IgG, (anti-idiotypic        scFv 4.15.64—MK062 Matriptase site—CD3—N-terminal fused to CD3        Fab—inert Fc) with N-terminal fused anti CD3 scFv 4.15.64 and        protease-cleavable linker.    -   Identification No. 7860: monovalent CD3 IgG, (anti-idiotypic        scFv 4.32.63—MK062 Matriptase site—CD3—N-terminal fused to CD3        Fab—inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and        protease-cleavable linker.    -   Identification No. 7857: monovalent CD3 IgG, (anti-idiotypic        scFv 4.15.64—non-cleavable linker—CD3—N-terminal fused to CD3        Fab—inert Fc) with N-terminal fused anti CD3 scFv 4.15.64 and        protease-cleavable linker.    -   Identification No. 7858: monovalent CD3 IgG, (anti-idiotypic        scFv 4.32.63—non-cleavable linker—CD3—N-terminal fused to CD3        Fab—inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and        protease-cleavable linker.    -   Identification No. 7861: monovalent CD3 IgG, (CD3 Fab—inert Fc)        with N-terminal fused anti CD3 scFv 4.15.64/4.32.63 and protease        linker.

Anti-idiotypic (ID) binder sequences were obtained by RACE-PCR (rapidamplification of cDNA ends) from RNA of Hybridoma cells. Hybridoma cellswere obtained by immunization of mice with CH2527(VL_7-46(13)/VH_3-23(12)) Fab-fragment. Single chain Fv (ScFv) sequencesynthesis was ordered from Invitrogen including the necessaryrestriction sites for cloning. Six different anti-idiotypic CH2527binders were compared for their affinities (FIG. 2 , resultBiacore-Analytics (AG M. Schräml) at 25° C./37° C. (Analyt:MAK<CEA/CD3>rH)) and two of them were cloned as N-terminal fusions atthe heavy chain of CD3 Fab-Fc.

The anti-ID single chain Fv DNA sequences were subcloned in frame withthe CD3 VH chain pre-inserted into the respective recipient mammalianexpression vector. Protein expression is driven by an MPSV promoter anda synthetic polyA signal sequence is present at the 3′ end of the CDS.In addition each vector contains an EBV OriP sequence.

The molecules were produced by co-transfecting HEK293-EBNA cells growingin suspension with the mammalian expression vectors usingpolyethylenimine (PEI). The cells were transfected with thecorresponding expression vectors in a 1:1:2 ratio (“Fc hole (CH2-CH3)”:“common light chain (CLC)”: “vector heavy chain knob(scFv-VH-CH1-CH2-CH3)”).

For transfection, HEK293 EBNA cells were cultivated in serum free ExCellculture medium containing 6 mM L-glutamine and 250 mg/l G418. For theproduction in 600 ml tubespin flasks (max. working volume 400 mL) 800million HEK293 EBNA cells were seeded 24 hours before transfectionwithout G418. For transfection 800 mio cells were centrifuged for 5 minat 210× g and supernatant was replaced by 40 ml pre-warmed CD CHO mediumcontaining 6 mM L-Glutamine. Expression vectors were mixed with 40 ml CDCHO medium containing 6 mM L-Glutamine to a total amount of 400 μg DNA.After addition of 1080 μl PEI solution (2.7 μg/ml) the mixture wasvortexed for 15 s and subsequently incubated for 10 min at roomtemperature. Afterwards cells were mixed with the DNA/PEI solution,transferred to a 600 ml tubespin flask and incubated for 3 hours at 37°C. in an incubator with a 5% CO₂ atmosphere. After incubation, 320 mlExCell+6 mM L-glutamine+5 g/L Pepsoy+1.0 mM VPA+3 g/l glucose medium wasadded and cells were cultivated for 24 hours prior to feeding with 7%Feed 7. After 6-7 days cultivation supernatant was collected forpurification by centrifugation for 20-30 min at 210× g (Sigma 8Kcentrifuge). The solution was sterile filtered (0.22 μm filter) andsodium azide in a final concentration of 0.01% w/v was added. Thesolution was kept at 4° C. until purification. The secreted protein waspurified from cell culture supernatants by affinity chromatography usingProteinA affinity chromatography, followed by one to two size exclusionchromatographic steps.

For affinity chromatography supernatant was loaded on a HiTrap Protein AFF column (CV=5 mL, GE Healthcare) equilibrated with 25 ml 20 mM sodiumphosphate, 20 mM sodium citrate, 0.5M sodium chloride, 0.01% Tween-20 pH7.5. Unbound protein was removed by washing with at least 10 columnvolumes 20 mM sodium phosphate, 20 mM sodium citrate, 0.5M sodiumchloride, 0.01% Tween-20 pH 7.5 and target protein was eluted in 20column volumes (gradient from 0%-100%) 20 mM sodium citrate, 0.5M sodiumchloride, 0.01% Tween-20 pH 2.5. Protein solution was neutralized byadding 1/10 of 2 M Tris pH 10.5. Target protein was concentrated withAmicon®Ultra-15 Ultracel 30K (Merck Millipore Ltd.) to a volume of 4 mlmaximum prior loading on a HiLoad Superdex 200 column (GE Healthcare)equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0, 0.01%Tween20.

For analytics after size exclusion chromatography the purity andmolecular weight of the molecules in the single fractions were analyzedby SDS-PAGE in the absence of a reducing agent and staining withCoomassie (InstantBlue™, Expedeon). The NuPAGE® Pre-Cast gel system(4-12% Bis-Tris, Invitrogen or 3-8% Tris-Acetate, Invitrogen) was usedaccording to the manufacturer's instruction.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm divided by the molarextinction coefficient calculated on the basis of the amino acidsequence.

Purity and molecular weight of the molecules after the finalpurification step were analyzed by CE-SDS analyses in the presence andabsence of a reducing agent. The Caliper LabChip GXII system (CaliperLifescience) was used according to the manufacturer's instruction. Theaggregate content of the molecules was analyzed using a TSKgel G3000 SWXL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mMNaCl, 200 mM L-arginine monohydrocloride, 0.02% (w/v) NaN3, pH 6.7running buffer at 25° C. The final quality of all molecules was good,with ≥92% monomer content.

TABLE 2 Summary of production and purification of protease activatedmonovalent CD3 IgG molecules. Analytical SEC Titer Yield(HMW/Monomer/LMW) Molecule [mg/l] [mg/l] [%] 1 12 3.38 2.21/95.5/2.29 29 1.75 4.86/95.14/0 3 15 4.8 6.93/93.07/0 4 4.5 0.26 4.88/95.12/0 5105.3 26.3 0/100/0

Example 2 Cleavage and Stability of Protease Activated IgGs

Capillary Electrophoresis of protease activated IgG molecules.Comparison of untreated sample and treated sample showed that theanti-ID scFv was completely cleaved off after treatment withrhMatriptase/ST14 (R&D Systems) indicated by the size shift in the SDSpage analysis (FIG. 3 ). Analysis of samples incubated for 48 h at 37°C. confirmed stability of the molecules in formulation buffer (FIG.3A-D).

Example 3 Masking Effect of Anti-Idiotypic scFv for CD3 IgG

The efficiency of masking the CD3 binder by N-terminal fusion of ananti-idiotypic CD3 scFv was shown by a Jurkat-NFAT reporter assay.Jurkat-NFAT reporter cells (a human acute lymphatic leukemia reportercell line with a NFAT promoter-regulated luciferase expression,GloResponse Jurkat NFAT-RE-luc2P, Promega #CS176501) express activefirefly luciferase if the NFAT promoter is activated by binding of CD3c.The intensity of the luminescence signal upon addition of luciferasesubstrate is proportional to the intensity of CD3 activation andsignaling. Completely unmasked monovalent CD3 molecules served as apositive control. The treatment was done with rhMatriptase/ST14 (R&DSystems) for 48 h at 37° C. In parallel Bug/ml Anti human Fc Antibody(BioLegends) were coated in 0.025 ul/well PBS for 48 h at 4° C. inwhite-walled, clear bottom 96-well (flat)-plate (Greiner Bio-One). PBSwas removed by pipetting before monovalent IgGs were added at theindicated concentration range of 200 nM-2.56 pM. Plates were incubatedfor about 30 min at 4° C. Subsequently, Jurkat-NFAT reporter cells wereharvested and viability assessed using ViCell. Cells were resuspended inJurkat medium (RPMI1640, 2 g/1 Glucose, 2 g/l NaHCO₃, 10% FCS, 25 mMHEPES, 2 mM L-Glutamin, 1× NEAA, 1× Sodium-pyruvate) without Hygromycineand 100 μl per well (25.000 cells/well) were added to the crosslinkedmonovalent CD3 IgGs. Cells were incubated for 3 h at 37° C. in ahumidified incubator. Plates were taken out of the incubator for about10 min to adapt to room temperature prior to Luminescence read out. 100μl/well of ONE-Glo solution (1:1 ONE-Glo and assay medium volume perwell) were added to wells and incubated for 10 min at room temperaturein the dark. Luminescence was detected using WALLAC Victor3 ELISA reader(PerkinElmer2030), 1 sec/well as detection time. 7857 (4.15.64 mask withnon-cleavable linker) and 7859 (untreated) show significantly reducedCD3c binding compared to unmasked (7861) and pretreated molecule (7859treated) (FIG. 4A). 7760 was included as a control to show thatN-terminal linkage does not block CD3 binding itself. 7858 (4.32.63 maskwith non-cleavable linker) and 7860 (untreated) show significantlyreduced CD3c binding compared to unmasked (7861) and pretreated molecule(7860 treated) (FIG. 4B). In line with the affinities of theanti-idiotypic CD3 binders the 4.32.63 mask is much more efficient thanthe 4.15.64. In terms of EC50 values (FIG. 4C) the 4.32.63 masked CD3binder binds 54 fold less than the unmasked CD3 binder 7861. For the4.15.64 mask it is only 16 fold less binding than for 7861. Depending onthe tumor target and the target binder the best mask can be evaluated.

Example 4 Preparation of Anti FolR1/Anti-CD3 T Cell Bispecific (TCB)Molecules with Anti CD3 scFv

Several T cell bispecific (TCB) molecules with various anti-idiotypicscFv were produced and are schematically depicted in FIGS. 5A-5H withtheir respective ID number. The following molecules were prepared:

-   -   ID7344: FolR1 16D5 2+1 IgG, classic format (anti-idiotypic scFv        4.15.64—MK062 Matriptase site—CD3—N-terminal fused to FolR1        VH—inert Fc) with N-terminal fused anti CD3 scFv 4.15.64 and        protease linker (FIG. 5A, SEQ ID NOs 1, 2 and 3).    -   ID7496: FolR1 16D5 2+1 IgG, classic format (anti-idiotypic scFv        4.32.63—MK062 Matriptase site—CD3—N-terminal fused to FolR1        VH—inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and        protease linker (FIG. 5C, SEQ ID NOs 1, 3 and 4).    -   ID7676: FolR1 16D5 2+1 IgG, classic format (anti-idiotypic scFv        4.15.64—non-cleavable GS linker—CD3—N-terminal fused to FolR1        VH—inert Fc) with N-terminal fused anti CD3 scFv 4.15.64 and        protease linker (FIG. 5B, SEQ ID NOs 1, 3 and 6).    -   ID7611: FolR1 16D5 2+1 IgG, classic format (anti-idiotypic scFv        4.32.63—non-cleavable GS linker—CD3—N-terminal fused to FolR1        VH—inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and        protease linker (FIG. 5D, SEQ ID NOs 1, 3 and 5).

Anti-idiotypic (ID) binder sequences were obtained by RACE-PCR (rapidamplification of cDNA ends) from RNA of Hybridoma cells. Hybridoma cellswere obtained by immunization of mice. Single chain Fv (ScFv) sequencesynthesis was ordered at Invitrogen including the necessary restrictionsites for cloning. Six different anti-idiotypic CH2527 binders werecompared for their affinities (FIG. 2 , result Biacore-Analytics (AG M.Schräml) at 25° C./37° C. (Analyt: MAK<CEA/CD3>rH)) and four of themwere cloned as N-terminal fusions at the HC of CD3-FolR1 16D5 TCB.

The anti-ID single chain Fv DNA sequences were subcloned in frame withthe CD3 VH chain pre-inserted into the respective recipient mammalianexpression vector. Protein expression is driven by an MPSV promoter anda synthetic polyA signal sequence is present at the 3′ end of the codingsequence (CDS). In addition each vector contains an EBV OriP sequence.

The molecules were produced by co-transfecting HEK293-EBNA cells growingin suspension with the mammalian expression vectors usingpolyethylenimine (PEI). The cells were transfected with thecorresponding expression vectors in a 1:3:2 ratio (“vector heavy chainhole (VH-CH1-CH2-CH3)”: “common light chain (CLC)”: “vector heavy chainknob (scFv-VH-CH1-VH-CH1-CH2-CH3)”).

For transfection HEK293 EBNA cells were cultivated in serum free ExCellculture medium containing 6 mM L-glutamine and 250 mg/l G418. For theproduction in 600 ml tubespin flasks (max. working volume 400 mL) 800million HEK293 EBNA cells were seeded 24 hours before transfectionwithout G418. For transfection 800 mio cells were centrifuged for 5 minat 210× g and supernatant was replaced by 40 ml pre-warmed CD CHO mediumcontaining 6 mM L-Glutamine. Expression vectors were mixed with 40 ml CDCHO medium containing 6 mM L-Glutamine to a total amount of 400 μg DNA.After addition of 1080 μl PEI solution (2.7 μg/ml) the mixture wasvortexed for 15 s and subsequently incubated for 10 min at roomtemperature. Afterwards cells were mixed with the DNA/PEI solution,transferred to a 600 ml tubespin flask and incubated for 3 hours at 37°C. in an incubator with a 5% CO₂ atmosphere. After incubation, 320 mlExCell+6 mM L-glutamine+5 g/L Pepsoy+1.0 mM VPA+3 g/l glucose medium wasadded and cells were cultivated for 24 hours prior to feeding with 7%Feed 7. After 6-7 days cultivation supernatant was collected forpurification by centrifugation for 20-30 min at 210× g (Sigma 8Kcentrifuge). The solution was sterile filtered (0.22 μm filter) andsodium azide in a final concentration of 0.01% w/v was added. Thesolution was kept at 4° C. until purification.

The secreted protein was purified from cell culture supernatants byaffinity chromatography using ProteinA affinity chromatography, followedby one to two size exclusion chromatographic steps.

For affinity chromatography supernatant was loaded on a HiTrap Protein AFF column (CV=5 mL, GE Healthcare) equilibrated with 25 ml 20 mM sodiumphosphate, 20 mM sodium citrate, 0.5M sodium chloride, 0.01% Tween-20 pH7.5. Unbound protein was removed by washing with at least 10 columnvolumes 20 mM sodium phosphate, 20 mM sodium citrate, 0.5M sodiumchloride, 0.01% Tween-20 pH 7.5 and target protein was eluted in 20column volumes (gradient from 0%-100%) 20 mM sodium citrate, 0.5M sodiumchloride, 0.01% Tween-20 pH 2.5. Protein solution was neutralized byadding 1/10 of 2 M Tris pH 10.5. Target protein was concentrated withAmicon®Ultra-15 Ultracel 30K (Merck Millipore Ltd.) to a volume of 4 mlmaximum prior loading on a HiLoad Superdex 200 column (GE Healthcare)equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0, 0.01%Tween20.

For analytics after size exclusion chromatography the purity andmolecular weight of the molecules in the single fractions were analyzedby SDS-PAGE in the absence of a reducing agent and staining withCoomassie (InstantBlue™, Expedeon). The NuPAGE® Pre-Cast gel system(4-12% Bis-Tris, Invitrogen or 3-8% Tris-Acetate, Invitrogen) was usedaccording to the manufacturer's instruction.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm divided by the molarextinction coefficient calculated on the basis of the amino acidsequence.

Purity and molecular weight of the molecules after the finalpurification step were analyzed by CE-SDS analyses in the presence andabsence of a reducing agent. The Caliper LabChip GXII system (CaliperLifescience) was used according to the manufacturer's instruction.

The aggregate content of the molecules was analyzed using a TSKgel G3000SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mMNaCl, 200 mM L-arginine monohydrocloride, 0.02% (w/v) NaN3, pH 6.7running buffer at 25° C. The final quality of all molecules was good,with ≥92% monomer content.

TABLE 2 Summary of production and purification of protease activated TCBmolecules. Analytical SEC Titer Yield (HMW/Monomer/LMW) Molecule [mg/l][mg/l] [%] 1 33 3.7 0.98/92.7/6.32 2 11 0.55 3.76/96.24/0 3 12.9 0.892.9/93.82/2.19 4 6.7 0.35 4.59/95.41/0

Example 5 Transient Expression of Protease Activated TCBs

Different plasmid ratios used for transfection were compared by sizeexclusion chromatography as the knob chain was suspected to be expressedin lower levels compared to the hole chain and the light chain. As shownin FIGS. 6 and 7 , using a plasmid ratio of 1(hole): 2 (knob): 3 (CLC)(FIG. 7 ) instead of 1(hole): 1 (knob): 3 (CLC) (FIG. 6 ) increased theyield of correct molecule (left peak) and decreased the amount of holehole homodimers (right peak).

Example 6 Cleavage and Stability of Protease Activated TCB

Protease activated TCBs were analyzed by capillary electrophoresis.Comparison of untreated sample and treated sample showed that theanti-idiotype scFc moiety was completely cleaved off after treatmentwith rhMatriptase/ST14. Analysis of samples incubated for 48 h at 37° C.confirmed stability of the molecules in formulation buffer (FIGS.12A-12D).

Example 7 Cell Killing Using Target Cell Lines that Express DifferentLevels of FolR1

T-cell-mediated cell killing induced by protease activated TCB moleculeswas assessed using target cell lines expressing different levels ofFolR1 (FIG. 13 ). Human PBMCs were used as effector cells and cellkilling was detected at 48 h of incubation with the protease activatedTCB molecules. Human Peripheral blood mononuclear cells (PBMCs) wereisolated from fresh taken blood or from buffy coats obtained fromhealthy human donors. For fresh blood 50 ml Leucosep tubes(GreinerBioOne) were used for preparation. For enriched lymphocytepreparations (buffy coats) Histopaque-1077 density preparation was used.Blood/buffy coat was diluted 1:1 with sterile PBS and layered overHistopaque gradient (Sigma, #H8889). After centrifugation (450×g, 30minutes, w/o break, room temperature), the plasma above thePBMC-containing interphase was discarded and PBMCs transferred in a newfalcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400× g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet resuspended in 2 ml ACK buffer forErythrocytes lysis. After incubation at 37° C. for about 2 −3 minutesthe tubes were filled with sterile PBS to 50 ml and centrifuged at 350×gfor 10 minutes. This washing step was repeated once prior toresuspension of PBMCs in RPMI1640 medium containing 2% FCS and 1×GlutaMax at 37° C., 5% CO₂ in cell incubator until further use. Briefly,adherent target cells were harvested with Trypsin/EDTA, counted, checkedfor viability and resuspended at 0.4×10⁶ cells/ml in assay medium(RPMI1640, 2% FCS, 1× GlutaMax). Target cells were plated at a densityof 20 000 cells/well using round-bottom 96-well plates. For the killingassay, the molecules were added at the indicated concentrations intriplicates. FolR1 16D5 TCB was included as positive control and anuntargeted TCB molecule (binding to CD3 but not to a target cellantigen) was included as negative control. PBMCs were added to targetcells at final E:T ratio of 10:1. Target cell killing was assessed after48 h of incubation at 37° C., 5% CO₂ by quantification of LDH releaseinto cell supernatants by apoptotic/necrotic cells (LDH detection kit,Roche Applied Science, #11 644 793 001). Maximal lysis of the targetcells (=100%) was achieved by incubation of target cells with 1% TritonX-100 1 h before LDH readout. Minimal lysis (=0%) refers to target cellsco-incubated with effector cells without any TCB.

The results (FIGS. 14A, 15A, 16, 17, 18A, 19A and 20A) show that theprotease activated TCB with anti-idiotypic CD3 scFv moiety N-terminallylinked by a non-cleavable linker (#7676 and #7611, FIGS. 5B and D,respectively) were able to significantly reduce cell lysis on Skov3 andHT29 cells. #7611 (FIG. 5D) led to reduced killing on Hela cells whileanti-idiotypic CD3 scFv 4.15.64 in #7676 (FIG. 5B) was less efficient inreduction of cell lysis. This is in line with affinities of theanti-idiotypic CD3 scFv N moiety. The higher affinity scFv moiety masksmore efficiently.

Comparable potency of treated and untreated TCBs suggests Matriptaseexpression of Hela and Skov3 cells. Expression of Matriptase seems to belower in HT29 cells. Treatment of Mkn-45, a FolR1 negative cell line,shows only weak killing with all molecules used herein (FIG. 15A).

Example 8 T-Cell Activation after Co-Incubation of Tumor Cell Lines withHuman PBMCs

T-cell activation mediated by protease activated TCB molecules wasassessed on Hela, Skov3 and HT29 cells. Human PBMCs were used aseffector cells and the T cell activation was detected at 48 h ofincubation with target cells and the antibodies. Target cells wereplated at a density of 20 000 cells/well using round-bottom 96-wellplates. Molecules were added at the indicated concentrations intriplicates. FolR1 16D5 TCB was included as positive control and anuntargeted TCB molecule (binding to CD3 but not to a target cellantigen) was included as negative control. PBMCs were added to targetcells at final E:T ratio of 10:1. T-cell activation was assessed after48 h of incubation at 37° C., 5% CO₂ by quantification of CD25 and CD69on CD4 positive and CD8 positive T cells. T cell activation results areconsistent with the results observed in the previous example assessingtarget cell killing (Example 7).

Example 9 T-Cell Activation Mediated by Protease-Activated TCBs andTarget Cell Lines Expressing Low Antigen Levels

T-cell activation mediated by protease activated TCB molecules wasassessed on HT29 cells expressing only low levels of FolR1 (FIG. 13 ).Human PBMCs isolated from buffy coat were used as effector cells. Forenriched lymphocyte preparations (buffy coats) Histopaque-1077 densitypreparation was used. Buffy coat was diluted 1:1 with sterile PBS andlayered over Histopaque gradient (Sigma, #H8889). After centrifugation(450×g, 30 minutes, w/o break, room temperature), the plasma above thePBMC-containing interphase was discarded and PBMCs transferred in a newfalcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400× g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet resuspended in 2 ml ACK buffer forErythrocytes lysis. After incubation at 37° C. for about 2 −3 minutesthe tubes were filled with sterile PBS to 50 ml and centrifuged at 350×gfor 10 minutes. This washing step was repeated once prior toresuspension of PBMCs in RPMI1640 medium containing 2% FCS and1×GlutaMax at 37° C., 5% CO₂ in cell incubator until further use.Briefly, adherent target cells were harvested with Trypsin/EDTA,counted, assessed for viability and resuspended at 0.4×10⁶ cells/ml inassay medium (RPMI1640, 2% FCS, 1× GlutaMax). Target cells were platedat a density of 20 000 cells/well using round-bottom 96-well plates.Molecules were added at the indicated concentrations in triplicates.FolR1 16D5 TCB was included as positive control and an untargeted TCBmolecule (binding to CD3 but not to a target cell antigen) was includedas negative control. PBMCs were added to target cells at final E:T ratioof 10:1. T-cell activation was assessed after 48 h of incubation at 37°C., 5% CO₂ by quantification of CD25 and CD69 on CD4 positive andCD8-positive T cells. The potency of treated protease activated TCB iscomparable to 16D5 TCB (6298). The 16D5 TCB (inverted format) showhigher potency than the classic format. Masked TCBs with non-cleavablelinker or without Matriptase pre-treatment do not induce T cellactivation on this cell line. For cell lines with low or medium FolR1expression levels both anti-idiotypic scFvs are sufficient in maskingthe CD3 Fab (FIGS. 22A and B).

Example 10 T-cell activation mediated by protease activated TCB withprimary cell line HRCEpiC

T-cell activation mediated by protease activated TCB molecules wasassessed on primary Human Renal Cortical Epithelial Cell (ScienceCell)cells expressing only very little amounts of FolR1 (FIG. 13 ). HumanPBMCs isolated from buffy coat were used as effector cells. For enrichedlymphocyte preparations (buffy coats) Histopaque-1077 densitypreparation was used. Buffy coat was diluted 1:1 with sterile PBS andlayered over Histopaque gradient (Sigma, #H8889). After centrifugation(450×g, 30 minutes, without break at room temperature), the plasma abovethe PBMC-containing interphase was discarded and PBMCs transferred in anew falcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400× g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet resuspended in 2 ml ACK buffer forErythrocytes lysis. After incubation at 37° C. for about 2 −3 minutesthe tubes were filled with sterile PBS to 50 ml and centrifuged at 350×gfor 10 minutes. This washing step was repeated once prior toresuspension of PBMCs in RPMI1640 medium containing 2% FCS and 1×GlutaMax at 37° C., 5% CO₂ in cell incubator until further use. Briefly,adherent target cells were harvested with Trypsin/EDTA, counted, checkedfor viability and resuspended at 0.4×10⁶ cells/ml in assay medium(RPMI1640, 2% FCS, 1× GlutaMax). Target cells were plated at a densityof 20 000 cells/well using round-bottom 96-well plates. Proteaseactivatable TCB molecules were added at the indicated concentrations intriplicates. FolR1 16D5 TCB was included as positive control and anuntargeted TCB molecule (binding to CD3 but not to a target cellantigen) was included as negative control. PBMCs were added to targetcells at final E:T ratio of 10:1. T-cell activation was assessed after48 h of incubation at 37° C., 5% CO₂ by quantification of CD25 and CD69on CD4 positive and CD8 positive T cells. Masked 16D5 TCB does notinduce T cell activation upon incubation with primary human renalcortical epithelial cells despite low level FolR1 expression at thehighest concentration of 10.000 pM of TCB, demonstrating theeffectiveness of the anti-idiotype masking moiety. Little T cellactivation can be observed for the 16D5 TCBs (inverted and classicformat) (FIG. 23 ).

Example 11 Anti-ID CD3 Fab Masking CD3 Binder of 16D5 TCB. Killing onOvcar3 Cells

T-cell-mediated target cell killing mediated by protease activated TCBmolecules was assessed on OVCAR3 cells (FIG. 24 ). Human PBMCs were usedas effector cells and cell killing was detected at 48 h of incubationwith the molecules. Human Peripheral blood mononuclear cells (PBMCs)were isolated from fresh taken blood of a healthy donor. 50 ml Leucoseptubes (GreinerBioOne) were used for preparation. Blood was diluted 1:1with sterile PBS and layered over Histopaque gradient (Sigma, #H8889).After centrifugation (450×g, 30 minutes, w/o break, room temperature),the plasma above the PBMC-containing interphase was discarded and PBMCstransferred in a new falcon tube subsequently filled with 50 ml of PBS.The mixture was centrifuged (400× g, 10 minutes, room temperature), thesupernatant discarded and the PBMC pellet resuspended in 2 ml ACK bufferfor Erythrocytes lysis. After incubation at 37° C. for about 2 −3minutes the tubes were filled with sterile PBS to 50 ml and centrifugedat 350×g for 10 minutes. This washing step was repeated once prior toresuspension of PBMCs in RPMI1640 medium containing 2% FCS and1×GlutaMax at 37° C., 5% CO₂ in cell incubator until further use.Briefly, adherent target cells were harvested with Trypsin/EDTA,counted, checked for viability and resuspended at 0.4×10⁶ cells/ml inassay medium (RPMI1640, 2% FCS, 1× GlutaMax). Target cells were platedat a density of 20 000 cells/well using round-bottom 96-well plates. Forthe killing assay, the molecules were added at the indicatedconcentrations in triplicates. FolR1 16D5 TCB was included as positivecontrol and an untargeted TCB molecule (binding to CD3 but not to atarget cell antigen) was included as negative control. PBMCs were addedto target cells at final E:T ratio of 10:1. Target cell killing wasassessed after 48 h of incubation at 37° C., 5% CO₂ by quantification ofLDH release into cell supernatants by apoptotic/necrotic cells (LDHdetection kit, Roche Applied Science, #11 644 793 001). Maximal lysis ofthe target cells (=100%) was achieved by incubation of target cells andPBMCs with 1% Triton X-100 1 h before LDH readout. Minimal lysis (=0%)refers to target cells co-incubated with effector cells without any TCB.The result (FIG. 24 ) shows that protease activated TCB withanti-idiotypic CD3 4.15.64 crossed Fab N—terminally linked by anon-cleavable linker is not significantly masking the CD3 binder.Further, Ovcar3 cells appear to express Matriptase because untreatedmolecule also induces killing of these cells.

Example 12 Killing on Skov3 and HeLa Cells with Three Different HumanPBMC Donors

T-cell killing mediated by protease activated TCB molecules was assessedon two different cell lines expressing different levels of FolR1 (FIGS.25-27 ). Human PBMCs were used as effector cells and cell killing wasdetected at 48 h of incubation with the molecules. Human Peripheralblood mononuclear cells (PBMCs) were isolated from buffy coats obtainedfrom healthy human donors. For enriched lymphocyte preparations (buffycoats) Histopaque-1077 density preparation was used. Blood/buffy coatwas diluted 1:1 with sterile PBS and layered over Histopaque gradient(Sigma, #H8889). After centrifugation (450×g, 30 minutes, w/o break,room temperature), the plasma above the PBMC-containing interphase wasdiscarded and PBMCs transferred in a new falcon tube subsequently filledwith 50 ml of PBS. The mixture was centrifuged (400× g, 10 minutes, roomtemperature), the supernatant discarded and the PBMC pellet resuspendedin 2 ml ACK buffer for Erythrocytes lysis. After incubation at 37° C.for about 2-3 minutes the tubes were filled with sterile PBS to 50 mland centrifuged at 350×g for 10 minutes. This washing step was repeatedonce prior to resuspension of PBMCs in RPMI1640 medium containing 10%FCS and 1× GlutaMax. PBMCs were resuspended in RPMI1640 mediumcontaining 10% FCS, 1× GlutaMax and 10% DMSO. PBMCs were frozenovernight at −80° C. in Cool Cell boxes before they were transferred toliquid nitrogen. 24 h before assay start, PBMCs were thawed and kept inRPMI1640 medium containing 10% FCS and 1× GlutaMax at 37° C., 5% CO2 incell incubator. The day before assay start adherent target cells wereharvested with Trypsin/EDTA, counted, checked for viability andresuspended at 0.4×10⁶ cells/ml in appropriate medium. Target cells wereplated at a density of 20 000 cells/well using flat-bottom 96-wellplates. On the day of assay start PBMCs were counted and checked forviability. PBMCs were centrifuged at 350 g for 5 min and resupsended inassay medium (RPMI1640, 2% FCS, 1× GlutaMax). The medium of target cellswas removed and PBMCs were added to the target cells before dilutedantibodies were added at the indicated concentrations in triplicates.FolR1 16D5 TCB was included as positive control and an untargeted TCBmolecule (binding to CD3 but not to a target cell antigen) was includedas negative control. PBMCs were added to target cells at E:T ratio of10:1. Target cell killing was assessed after 48 h of incubation at 37°C., 5% CO₂ by quantification of LDH release into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100 2 h before LDH readout.Minimal lysis (=0%) refers to target cells co-incubated with effectorcells without any TCB.

The results (FIGS. 25-27 ) show that FolR1 TCB with scFv 4.32.63N-terminally linked by a non-cleavable linker (FIG. 5D) induced reducedkilling on Hela cells at concentration of 100 pM and on Skov3 cells at aconcentration of 10 nM. FolR1 TCB with scFv 4.15.64 N-terminally linkedby a non-cleavable linker (FIG. 5B) was less efficient in reducingkilling on Skov3 cells at a concentration of 10 nM. The stronger mask,meaning the anti-idiotypic scFv 4.32.63 with the higher affinity, ismore efficient in masking the CD3 binder than the weak anti-idiotypicscFv 4.15.64. Comparable potency of treated and untreated TCBs suggestsprotease, e.g. Matriptase, expression by Hela and Skov3 cells.

Example 13 Preparation of the HER1 Binding Antibody GA201 Masked with anAnti-Idiotype GA201 scFv

The following molecules were prepared in this example:

-   -   1: GA201 IgG1 antibody with N-terminal fusion of an        anti-idiotypic GA201 scFv and Matrix Metalloprotease site in        glycine serine linker (SEQ ID NOs 32 and 34); and    -   2: HER1-binding IgG1 antibody GA201 (SEQ ID NOs 32 and 33).

Schematic illustrations thereof are shown in FIGS. 28 and 29 . The GA201anti-idiotypic (ID) binder sequence was obtained by RT-PCR (reversetranscription) from RNA of Hybridoma cells using degenerated primersbinding to the ends of the variable light and heavy chain, respectively.Hybridoma cells were obtained by immunization of mice. Single chain Fv(scFv) DNA sequence synthesis with flanking singular restrictionendonuclease sites was ordered at Geneart and cloned as N-terminalfusion at the GA201 light chain.

A Roche expression vector was used for the construction of all heavy andlight chain scFv fusion protein encoding expression plasmids. The vectoris composed of the following elements:

-   -   a hygromycin resistance gene as a selection marker,    -   an origin of replication, oriP, of Epstein-Barr virus (EBV),    -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli,    -   the immediate early enhancer and promoter from the human        cytomegalovirus (HCMV),    -   the human 1-immunoglobulin polyadenylation (“poly A”) signal        sequence, and    -   unique BamHI and XbaI restriction sites.

The molecules were produced by co-transfecting human embryonic kidney293-F cells growing in suspension with the mammalian expression vectorsusing the FreeStyle™ 293 Expression System according to themanufacturer's instruction (Invitrogen, USA). Briefly, suspensionFreeStyle™ 293-F cells were cultivated in FreeStyle™ 293 Expressionmedium at 37° C./8% CO₂ and the cells were seeded in fresh medium at adensity of 1-2×10⁶ viable cells/ml on the day of transfection.DNA-293fectin™ complexes were prepared in Opti-MEM I medium (Invitrogen,USA) using 325 μl of 293fectin™ (Invitrogen, Germany) and 250 μg ofheavy (“GA201 heavy chain”) and light chain (“anti-GA201 VH-VL scFv MMPcleavable linker G4S GA201 light chain” or “GA201 light chain”) plasmidDNA in a 1:1 molar ratio for a 250 ml final transfection volume.Antibody containing cell culture supernatants were harvested 7 daysafter transfection by centrifugation at 14000 g for 30 minutes andfiltered through a sterile filter (0.22 μm). Supernatants were stored at−20° C. until purification.

The secreted protein was purified from cell culture supernatants byaffinity chromatography using ProteinA affinity chromatography, followedby size exclusion chromatography. Briefly, sterile filtered cell culturesupernatants were applied to a HiTrap ProteinA HP (5 ml) columnequilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCland 2.7 mM KCl, pH 7.4). Unbound proteins were washed out withequilibration buffer. Antibody and antibody variants were eluted with0.1 M citrate buffer, pH 2.8, and the protein containing fractions wereneutralized with 0.1 ml 1 M Tris, pH 8.5. Then, the eluted proteinfractions were pooled, concentrated with an Amicon Ultra centrifugalfilter device (MWCO: 30 K, Millipore) to a volume of 3 ml and loaded ona Superdex200 HiLoad 120 ml 16/60 gel filtration column (GE Healthcare,Sweden) equilibrated with 20 mM Histidin, 140 mM NaCl, pH 6.0. Fractionscontaining purified GA201-anti-GA201-scFv or GA201 with less than 5%high molecular weight aggregates were pooled and stored as 1.0 mg/mlaliquots at −80° C.

For Protein analytics after size exclusion chromatography, the purityand molecular weight of the molecules in the single fractions wereanalyzed by SDS-PAGE in the absence of a reducing agent and stainingwith Coomassie (InstantBlue™, Expedeon). The NuPAGE® Pre-Cast gel system(4-12% Bis-Tris, Invitrogen or 3-8% Tris-Acetate, Invitrogen) was usedaccording to the manufacturer's instruction.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm divided by the molarextinction coefficient calculated on the basis of the amino acidsequence. Purity and molecular weight of the molecules after the finalpurification step were analyzed by CE-SDS analyses in the presence andabsence of a reducing agent. The Caliper LabChip GXII system (CaliperLifescience) was used according to the manufacturer's instruction. Theaggregate content of the molecules was analyzed by high-performance SECusing a Superdex 200 analytical size-exclusion column (GE Healthcare,Sweden) in 200 mM KH2PO4, 250 mM KCl, pH 7.0 running buffer at 25° C. 25μg protein were injected on the column at a flow rate of 0.5 ml/min andeluted isocratic over 50 minutes.

The final purity of all molecules was ≥95% monomer content as detectedby high performance SEC. The molecular weight of the anti-idiotypic scFvmasked GA201 was determined by CE-SDS analysis as 216.3 kDa under nonreducing conditions (FIG. 1A) and under reducing conditions as 58.3 kDafor the GA201 heavy chain and 60.3 kDa for the scFv linked GA201 lightchain (FIG. 30B), respectively. The molecular weight based on the aminoacid sequence was calculated as 49.2 kDa for the heavy chain and 51.9kDa for the scFv fused GA201 light chain, which indicates glycosylationof both chains in HEK293 cells.

TABLE 3 Summary of production and purification of protease-activatedGA201 IgG (FIG. 28) and GA201 (FIG. 29) control molecules. MoleculeSupernatant Protein A - Yield SEC -Yield 1 1.0 L 1.3 mg 0.4 mg 2 1.0 L26.4 mg   24 mg

Example 14 Masking Effect of an Anti-Idiotypic scFv for GA201 IgG

The efficiency of masking the HER1 binding of GA201 by N-terminal fusionof an anti-idiotypic GA201 scFv was shown by FACS analysis on HER1expressing H322M cells and Surface Plasmon Resonance (SPR) analysis on aHER1 coated chip surface. For proteolytic cleavage ofGA201-anti-GA201-scFv recombinant active human MMP2 (Calbiochem) wasused. 1 mg of GA201 anti-idiotypic scFv fused to GA201 by a glycineserine linker containing a MMP cleavage site was incubated with 1.2 μgMMP2 overnight at 37° C. in PBS.

For FACS analysis of HER1 binding of cleaved and uncleavedGA201-anti-GA201-scFv, the non-small cell lung cancer line H322M wasused. Cells were adjusted to 1×10⁶/ml and distributed to a 96-wellround-bottom plate. The molecules were added and incubated on ice for 30minutes. Cells were washed once with FACS buffer (PBS+2% FCS+0.1% sodiumazide) and re-suspended with a F(ab′)2-goat anti-human IgG Fc secondaryantibody FITC conjugate (ThermoFisher Scientific). After another 20minutes on ice, cells were washed twice and re-suspended in FACS bufferand analyzed in a BD FACS Canto II. 10000 cells were measured and themedian of the fluorescence signal was used for analysis. Before MMP-2cleavage of GA201-anti-GA201-scFv no binding to HER1 on H322M cells wasmeasurable, indicating complete masking of the GA201 binding domains bythe anti-idiotypic scFv (FIG. 31 ). Binding of uncleavedGA201-anti-GA201-scFv was comparable to an unspecific isotype IgGcontrol antibody (FIG. 31 ). In contrast, MMP cleavage of theanti-idiotypic scFv leads to activation of GA201 and binding to HER1 onH322M cells was restored to similar levels as the unmasked parentalantibody GA201 (FIG. 31 ) To confirm the FACS binding data of maskedGA201 binding after MMP cleavage, we also performed a SPR experiment assecond analytical method using a Biacore T100 instrument (GE HealthcareBiosciences AB, Uppsala, Sweden). HER1 was immobilized on the surface ofa CMS biosensorchip using standard amine-coupling chemistry. The HER1extracellular domain was injected in sodium acetate, pH 5.0 at 1 μg/ml.Reference control flow cells were treated in the same way but withvehicle buffer only. GA201-anti-GA201-scFv, before and after anovernight MMP cleavage, and GA201 were diluted in 1×PBS pH 7.4, 0.05%Tween20 Roche Diagnostics GmbH) and injected at increasingconcentrations between 3.125 and 50 nM with a flow rate of 30 μl/min.The association phase was 3 minutes and the dissociation time was 10minutes. HER1 binding was regenerated with an inject of 0.85% phosphoricacid for 30 s at a flow rate of 5 μl/min. Kinetic rate constants andequilibrium dissociation constants were calculated by using the 1:1Langmuir binding model within the Biaevaluation software. A K_(D) valueof 1 nM for binding of HER1 was determined for the GA201 parentalunmasked antibody (FIG. 32 ). After an overnight MMP-2 incubation ofGA201-anti-GA201-scFv, a K_(D) value of 2 nM was measured with similark_(a) and k_(d) rate constants for association and dissociation as theunmasked control antibody, indicating complete restoration of HER1binding by protease cleavage (FIG. 32 ). Uncleaved GA201-anti-GA201-scFvdid not show any binding to HER1 in SPR analysis (FIG. 32 ). In summary,we have demonstrated a complete loss of binding to HER1 by fusion of ananti-idiotypic scFv to the N-terminus of the IgG1 antibody GA201 withtwo independent analytical methods. Furthermore, binding to HER1 wasfully restored by removal of the scFv through protease cleavage in theMMP cleavage site in the glycine serine linker.

Example 15 Preparation of Anti FolR1/Anti-CD3 andantiMesothelin/Anti-CD3 T Cell Bispecific (TCB) Molecules with Anti CD3scFv

Several T cell bispecific (TCB) molecules with various anti-idiotypicscFv were produced and are schematically depicted in FIGS. 33A-33J withtheir respective ID number. The following molecules were prepared:

-   -   ID 8364: “FolR1 16D5 2+1 IgG, classic format (anti idiotypic        scFv 4.32.63—MMP9—MK062 Matriptase site—CD3—N-terminal fused to        FolR1 VH—inert Fc) with N-terminal fused anti CD3 scFv 4.32.63        and MMP9—MK062 protease linker” (FIG. 33A, SEQ ID NOs 1, 3 and        72).    -   ID 8363: “FolR1 16D5 2+1 IgG, classic format (anti idiotypic        scFv 4.32.63—Cathepsin S/B site—CD3—N-terminal fused to FolR1        VH—inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and        Cathepsin S/B protease linker” (FIG. 33B, SEQ ID NOs 1, 3 and        85).    -   ID 8365: “FolR1 16D5 2+1 IgG, inverted format, (anti idiotypic        scFv 4.32.63—MK062 Matriptase linker—CD3—N-terminal fused to CD3        VL—inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and        MK062 Matriptase linker” (FIG. 33C, SEQ ID NOs 1, 3, 73 and 74).    -   ID 8366: “FolR1 16D5 2+1 IgG, inverted format, (anti idiotypic        scFv 4.32.63—non-cleavable GS linker—CD3—N-terminal fused to CD3        VL—inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and        non-cleavable GS linker” (FIG. 33D).    -   ID 8672: “aMesothelin 2+1 IgG, classic format, MSLN charged        variants, CD3 crossed (anti idiotypic scFv 4.32.63—MMP9—MK062        Matriptase—CD3—N-terminal fused to aMesothelin VH—inert Fc) with        N-terminal fused anti CD3 scFv 4.32.63 and MMP9—MK062        Matriptase” (FIG. 33E, SEQ ID NOs 77, 78, 81, 82).    -   ID 8673: “aMesothelin 2+1 IgG, classic format, MSLN charged        variants, CD3 crossed (anti idiotypic scFv 4.32.63—non-cleavable        GS linker—CD3—N-terminal fused to aMesothelin VH—inert Fc) with        N-terminal fused anti CD3 scFv 4.32.63 non-cleavable GS linker”        (FIG. 33F).    -   ID 8674: “aMesothelin 2+1 IgG, inverted format, MSLN charged        variants, CD3 crossed (anti idiotypic scFv 4.32.63—MMP9—MK062        Matriptase—CD3—N-terminal fused to CD3 VH—inert Fc) with        N-terminal fused anti CD3 scFv 4.32.63 and MMP9—MK062        Matriptase” (FIG. 33G, SEQ ID NOs 76, 77, 78, 79).    -   ID 8675: “aMesothelin 2+1 IgG, inverted format, MSLN charged        variants, CD3 crossed (anti idiotypic scFv 4.32.63—non-cleavable        GS linker—CD3—N-terminal fused to CD3 VH—inert Fc) with        N-terminal fused anti CD3 scFv 4.32.63 and non-cleavable GS        linker” (FIG. 33H).    -   ID 8505: “aMesothelin 2+1 IgG, inverted format, MSLN charged        variants, CD3 (aMesothelin HC N-terminally fused to CD3 VL—inert        Fc)” (FIG. 33I).    -   ID 8676: “aMesothelin 2+1 IgG, classic format, MSLN charged        variants, CD3 crossed (aMesothelin IgG with CD3—N-terminal fused        to aMesothelin VH—inert Fc)” (FIG. 33J)

The variable domains were subcloned in frame with the pre-inserteddomains into the respective recipient mammalian expression vector.Protein expression is driven by an MPSV promoter and a synthetic polyAsignal sequence is present at the 3′ end of the CDS. In addition eachvector contains an EBV OriP sequence.

The molecules (except 8505, this molecule was produced byco-transfecting CHO cells growing in suspension with the mammalianexpression vectors. Transient transfection was done at Evitria AG(Switzerland).) were produced by co-transfecting HEK293-EBNA cellsgrowing in suspension with the mammalian expression vectors usingpolyethylenimine (PEI). For transfection HEK293 EBNA cells werecultivated in serum free ExCell culture medium containing 6 mML-glutamine and 250 mg/l G418. For the production in 600 ml tubespinflasks (max. working volume 400 ml) 800 million HEK293 EBNA cells wereseeded 24 hours before transfection without G418. For transfection 800mio cells were centrifuged for 5 min at 210× g and supernatant wasreplaced by 40 ml pre-warmed CD CHO medium containing 6 mM L-Glutamine.Expression vectors were mixed with 40 ml CD CHO medium containing 6 mML-Glutamine to a total amount of 400 μg DNA. After addition of 1080 μlPEI solution (2.7 μg/ml) the mixture was vortexed for 15 s andsubsequently incubated for 10 min at room temperature. Afterwards cellswere mixed with the DNA/PEI solution, transferred to a 600 ml tubespinflask and incubated for 3 hours at 37° C. in an incubator with a 5% CO2atmosphere. After incubation, 320 ml ExCell+6 mM L-glutamine+5 g/LPepsoy+1.0 mM VPA+3 g/l glucose medium was added and cells werecultivated for 24 hours prior to feeding with 7% Feed 7. After 6-7 daysthe cultivation supernatant was collected for purification bycentrifugation for 20-30 min at 210× g (Sigma 8K centrifuge). Thesolution was sterile filtered (0.22 μm filter) and sodium azide in afinal concentration of 0.01% w/v was added. The solution was kept at 4°C. until purification.

The secreted protein was purified from cell culture supernatants byaffinity chromatography using ProteinA affinity chromatography, followedby one to two size exclusion chromatographic steps.

For affinity chromatography supernatant was loaded on a Protein AMabSelectSure (CV=5 mL, GE Healthcare) equilibrated with 20 mM SodiumCitrate, 20 mM Sodium Phosphate, pH 7.5. Unbound protein was removed bywashing with at least 10 column volumes 20 mM Sodium Citrate, 20 mMSodium Phosphate, pH 7.5 and target protein was eluted in 20 columnvolumes (gradient from 0%-100%) 20 mM Sodium Citrate, 100 mM SodiumChloride, 100 mM Glycine, pH 3.0. Protein solution was neutralized byadding 1/10 of 0.5 M Na2HPO4 pH 8.0. Target protein was concentratedwith Amicon®Ultra-15 Ultracel 30K (Merck Millipore Ltd.) to a volume of4 ml maximum prior loading on a HiLoad Superdex 200 column (GEHealthcare) equilibrated with 20 mM Histidine, 140 mM NaCl, 0.01% TweenpH 6.0.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm divided by the molarextinction coefficient calculated on the basis of the amino acidsequence.

Purity and molecular weight of the molecules after the finalpurification step were analyzed by CE-SDS analyses in the presence andabsence of a reducing agent. The Caliper LabChip GXII system (CaliperLifescience) was used according to the manufacturer's instruction.

The aggregate content of the molecules was analyzed using a TSKgel G3000SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mMNaCl, 200 mM L-arginine monohydrocloride, 0.02% (w/v) NaN3, pH 6.7running buffer at 25° C.

The final quality of all molecules was good, with ≥95% monomer content.

TABLE 4 Summary of production and purification of protease activated TCBmolecules. Analytical SEC Titer Yield (HMW/Monomer/LMW) Molecule [mg/l][mg/l] [%] 1 (8364) 34.55 1.72 0.68/99.32/0 2 (8363) 33.75 1.594.02/95.98/0 3 (8365) 5.35 0.24 2.71/96.46/0.83 4 (8366) 4.2 0.434.908/96.02/0 5 (8672) 13.8 1.59 3.96/96.04/0 6 (8673) 14 1.992.15/97.85/0 7 (8674) 3.6 0.96 6.27/93.73/0 8 (8675) 5.2 0.595.81/90.63/3.57 9 (8505) 120 20.46 0.47/99.32/0.22 10 (8676)  22.5 3.841.98/96.21/1.81

Example 16 Quality Control and Stability—Capillary Electrophoresis SDSAnalysis of Different TCB molecules

Purity and molecular weight of the molecules after the finalpurification step were analyzed by CE-SDS analyses in the presence andabsence of a reducing agent. The Caliper LabChip GXII system (CaliperLifescience) was used according to the manufacturer's instruction.Comparison of untreated molecules (stored at 4° C.), treated molecules(treated with appropriate recombinant protease (R&D Systems) for 24 h at37° C. and molecule incubated for 72 h at 37° C. (FIGS. 34, 35A, and35B). Comparison of the untreated and treated molecule shows completecleavage of the anti ID scFv after rhMatriptase/ST14 treatment for theinverted format containing MK062 Matriptase linker but incompletecleavage of MMP9-MK062 Matriptase linker. rhCathepsin B and rhCathepsinS treatment is incomplete as well. The conditions for the purifiedenzymes have not been optimal.

Molecules incubated at 37° C. for 72 h are running on the same heightthan pure molecules suggesting that the molecules are stable at 37° C.for the time of in vitro assay duration. Pre-stained protein Marker Mark12 (Invitrogen) was used for estimation of correct molecule weight.

Example 17

Comparison of Different Linkers and Formats of Protease Activated FolR1TCBs

Jurkat NFAT activation assay. Jurkat NFAT activation assay forcomparison of different formats and linkers of protease activated TCB.Jurkat-NFAT reporter cell line (Promega) is a human acute lymphaticleukemia reporter cell line with a NFAT promoter, expressing human CD3c.If the TCB binds the tumor target and the CD3 binder (crosslinkage)binds the CD3c Luciferase expression can be measured in Luminescenceafter addition of One-Glo substrate (Promega). 20.000 target cells wereseeded in 96-well white walled clear bottom plate (Greiner BioOne) in 50ul/well Jurkat medium (RPMI1640, 2 g/1 Glucose, 2 g/l NaHCO₃, 10% FCS,25 mM HEPES, 2 mM L-Glutamin, 1× NEAA, 1× Sodium-pyruvate) withoutHygromycine. Plates were incubated for about 20 hours at 37° C.Jurkat-NFAT reporter cells were harvested and viability was assessedusing ViCell. Cells were resuspended in Jurkat medium withoutHygromycine and 50 μl per well (50.000 cells/well) were added. The E:Tratio was 2.5:1 (based on cell number seeded). Antibodies were dilutedin Jurkat medium without Hygromycine and 50 ul/well were added. Cellswere incubated at 37° C. for 6 h in a humidified incubator before theywere taken out of the incubator for about 10 min to adapt to roomtemperature prior to Luminescence read out. 50 μl/well of ONE-Glosolution were added to wells and incubated for 10 min at roomtemperature in the dark. Luminescence was detected using WALLAC Victor3ELISA reader (PerkinElmer2030), 1 sec/well as detection time. Comparisonof the pretreated protease activated TCB (8364, grey filled squares) andFolR1 TCB (black triangles pointing down) showed that potency aftercleavage is recovered completely. No Luminescence was detectable forcells incubated with the masked TCB (containing a GS non-cleavablelinker, grey triangles pointing up) and the non-targeted TCB control(empty triangle pointing down) for both cell lines in this concentrationrange. The dotted line shows the Luminescence of target cells andeffector cells without any TCB (FIGS. 36A and 36B).

Example 18 Tumor Cell Cytotoxicity Mediated by Different Formats ofProtease Activated TCB

T-cell killing mediated by protease activated TCB molecules was assessedon cell lines expressing different levels of FolR1. Human Peripheralblood mononuclear cells (PBMCs) were isolated from buffy coats obtainedfrom healthy human donors. Buffy coat was diluted 1:1 with sterile PBSand layered over Histopaque gradient (Sigma, #H8889). Aftercentrifugation (450×g, 30 minutes, w/o break, room temperature) thePBMC-containing interphase was transferred in a new falcon tube that wassubsequently filled with 50 ml of PBS. The mixture was centrifuged (400×g, 10 minutes, room temperature), the supernatant was discarded and thePBMC pellet was resuspended in 2 ml ACK buffer for Erythrocytes lysis.After incubation for about 2-3 minutes at 37° C. the tubes were filledwith sterile PBS to 50 ml and centrifuged for 10 minutes at 350× g. Thiswashing step was repeated once prior to resuspension of PBMCs inRPMI1640 medium containing 10% FCS, 1× GlutaMax and 10% DMSO. PBMCs wereslowly frozen in CoolCell® Cell Freezing Containers (BioCision) at −80°C. and then transferred to liquid nitrogen. One day before assay startadherent target cells were harvested with Trypsin/EDTA, counted, checkedfor viability and resuspended in assay medium (RPMI1640, 2% FCS, 1×GlutaMax). Target cells were plated at a density of 20 000 cells/wellusing 96-well flat-bottom plates and incubated for about 20 h at 37° C.in a humidified incubator. About 20 h before assay start PBMCs werethawed in RPMI1640 medium (10% FCS, 1× GlutaMax). PBMCs were centrifugedat 350 g for 7 min. The pellet was resuspended in fresh medium(RPMI1640, 10% FCS, 1× GlutaMax) and incubated for max 24 h at 37° C. ina humidified incubator. On the day of the assay start PBMCs wereharvested and centrifuged at 350 g for 7 min. The pellet was resuspendedin assay medium and 0.2 mio PBMCs in 100 ul/well (E:T 10:1, based on thenumber of seeded target cells) were added to the target cells. Themolecules were diluted in assay medium (RPMI1640, 2% FCS, 1× GlutaMax)and 50 ul/well were added at the indicated concentrations in triplicatesbefore the plates were incubated for about 48 h at 37° C. in ahumidified incubator. Target cell killing was assessed after 48 h ofincubation at 37° C., 5% CO2 by quantification of LDH release into cellsupernatants by apoptotic/necrotic cells (LDH detection kit, RocheApplied Science, #11 644 793 001). Maximal lysis of the target cells(=100%) was achieved by incubation of target cells with 1% Triton X-10020 h before LDH readout. Minimal lysis (=0%) refers to target cellsco-incubated with effector cells without any TCB.

The results (FIGS. 37A and 37B) show the comparison of two differentformats of the Protease activated TCBs both containing the antiidiotypic CD3 scFv 4.32.63 linked with a MK062 Matriptase linker. Theinverted format of the protease activated TCB (8365, grey circles) seemsto be more potent in killing (HeLa and Skov-3 target cells) than theclassic format of the protease activated TCB (8408, dark grey trianglespointing up). However the inverted molecule containing the non-cleavablelinker (8366, light grey squares) is less efficient in masking than theclassic molecule (8409, dark grey triangles pointing down).

FIG. 37C HeLa target cell cytotoxicity. Comparison of classic Proteaseactivated TCB containing the anti idiotypic CD3 scFv 4.32.63 and GSlinkers with different protease sites. Protease activated TCB containingthe MMP9-Matriptase MK062 linker (8364, grey squares) reaches thepotency of FolR1 TCB (light grey triangles pointing down) whereas theprotease activated TCB containing only Matriptase MK062 (light greyrhomb) is less potent in killing HeLa cells. Molecules containingCathepsin site (grey circles) or non-cleavable linker (black trianglespointing down) are comparable. FIG. 37D Skov-3 target cell cytotoxicity.Comparison of classic Protease activated TCB containing the antiidiotypic CD3 scFv 4.32.63 and GS linkers with different protease sites.Protease activated TCB containing the MMP9-Matriptase MK062 linker(8364, grey squares) nearly reaches the potency of FolR1 TCB (light greytriangles pointing down) whereas the protease activated TCB containingonly Matriptase MK062 (light grey rhomb) is less potent in killingSkov-3 cells. The molecule containing Cathepsin site (grey circles) isless potent than the molecule containing only the Matriptase MK062 siteand the molecule containing the non-cleavable linker (black trianglespointing down) only induces killing below 10% in the indicatedconcentration range for Skov-3 cells.

Example 19

T-Cell Activation after Co-Incubation of Human Renal Epithelial CorticalCells or Human Bronchial Epithelial Cells with TCBs and Human PBMCs

T-cell activation mediated by protease activated TCB molecules wasassessed for HRCEpi (Human renal cortical epithelial cells) and HBEpiC(human bronchial epithelial cells expressing only little amounts ofFolR1. Human PBMCs were used as effector cells and T cell activationmarkers were stained after 48 h of incubation with the molecules andcells. Human Peripheral blood mononuclear cells (PBMCs) were isolatedfrom buffy coats obtained from healthy human donors. Buffy coat wasdiluted 1:1 with sterile PBS and layered over Histopaque gradient(Sigma, #H8889). After centrifugation (450×g, 30 minutes, w/o break,room temperature) the PBMC-containing interphase was transferred in anew falcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400× g, 10 minutes, room temperature), the supernatant wasdiscarded and the PBMC pellet was resuspended in 2 ml ACK buffer forErythrocytes lysis. After incubation for about two minutes at 37° C. thetubes were filled with sterile PBS to 50 ml and centrifuged for 10minutes at 350× g. This washing step was repeated once prior toresuspension of PBMCs in RPMI1640 medium containing 10% FCS, 1× GlutaMaxand 10% DMSO. PBMCs were slowly frozen in CoolCell® Cell FreezingContainers (BioCision) at −80° C. and then transferred to liquidnitrogen. One day before the assay was started adherent target cellswere harvested with Trypsin/EDTA, counted, checked for viability andresuspended in assay medium (RPMI1640, 2% FCS, 1× GlutaMax). Targetcells were plated at a density of 20 000 cells/well using 96-wellflat-bottom plates and incubated for about 20 h at 37° C. in ahumidified incubator. About 20 h before assay start PBMCs were thawed inRPMI1640 medium (10% FCS, 1× GlutaMax). PBMCs were centrifuged for 7 minat 350 g. The pellet was resuspended in fresh medium (RPMI1640, 10% FCS,1× GlutaMax) and incubated for max 24 h at 37° C. in a humidifiedincubator. On the day of the assay start PBMCs were harvested andcentrifuged for 7 min at 350 g. The pellet was resuspended in assaymedium and 0.2 mio PBMCs in 100 ul/well (E:T 10:1, based on the numberof seeded target cells) were added to the target cells. The moleculeswere diluted in assay medium (RPMI1640, 2% FCS, 1× GlutaMax) and addedat the indicated concentrations in triplicates before the plates wereincubated for about 48 h at 37° C. in a humidified incubator.

T-cell activation was assessed after 48 h of incubation at 37° C., 5%CO2 by quantification of CD25 and CD69 on CD4 positive and CD8 positiveT cells. FolR1 16D5 TCB (6298) and an untargeted TCB (binding to CD3 butnot to a target cell antigen, 7235) were included as controls. Eachpoint represents the mean value of triplicates of three different humanPBMC donors. Standard deviation is indicated in error bars. Unpaired ttest was used for statistical analysis. The results show an increase inCD69 for CD8 positive cells for the FolR1 TCB that is significantlyhigher than the median fluorescence intensity for the protease activatedTCBs (FIGS. 38A and 38B).

Example 20

Tumor Cell Cytotoxicity Mediated by Different Formats of ProteaseActivated Mesothelin (MSLN) TCB

T-cell killing mediated by protease activated TCB molecules was assessedon cell lines expressing different levels of Mesothelin (MSLN). HumanPeripheral blood mononuclear cells (PBMCs) were isolated from buffycoats obtained from healthy human donors. Buffy coat was diluted 1:1with sterile PBS and layered over Histopaque gradient (Sigma, #H8889).After centrifugation (450×g, 30 minutes, w/o break, room temperature)the PBMC-containing interphase was transferred in a new falcon tubesubsequently filled with 50 ml of PBS. The mixture was centrifuged (400×g, 10 minutes, room temperature), the supernatant was discarded and thePBMC pellet was resuspended in 2 ml ACK buffer for Erythrocytes lysis.After incubation for about two minutes at 37° C. the tubes were filledwith sterile PBS to 50 ml and centrifuged for 10 minutes at 350× g. Thiswashing step was repeated once prior to resuspension of PBMCs inRPMI1640 medium containing 10% FCS, 1× GlutaMax and 10% DMSO. PBMCs wereslowly frozen in CoolCell® Cell Freezing Containers (BioCision) at −80°C. and then transferred to liquid nitrogen. Adherent target cells wereharvested with Trypsin/EDTA, counted, checked for viability andresuspended in assay medium (RPMI1640, 2% FCS, 1× GlutaMax) one daybefore the assay was started. Target cells were plated at a density of20 000 cells/well using 96-well flat-bottom plates and incubated forabout 20 h at 37° C. in a humidified incubator. PBMCs were thawed inRPMI1640 medium (10% FCS, 1× GlutaMax) about 20 h before assay start.PBMCs were centrifuged for 7 min at 350 g. The pellet was resuspended infresh medium (RPMI1640, 10% FCS, 1× GlutaMax) and incubated for max 24 hat 37° C. in a humidified incubator. On the day of the assay start PBMCswere harvested and centrifuged for 7 min at 350 g. The pellet wasresuspended in assay medium and 0.2 mio PBMCs in 100 ul/well (E:T 10:1,based on the number of seeded target cells) were added to the targetcells. The molecules were diluted in assay medium (RPMI1640, 2% FCS, 1×GlutaMax) and added at the indicated concentrations in triplicatesbefore the plates were incubated for about 48 h at 37° C. in ahumidified incubator. Target cell killing was assessed after 48 h ofincubation at 37° C., 5% CO2 by quantification of LDH release into cellsupernatants by apoptotic/necrotic cells (LDH detection kit, RocheApplied Science, #11 644 793 001). Maximal lysis of the target cells(=100%) was achieved by incubation of target cells with 1% Triton X-10020 h before LDH readout. Minimal lysis (=0%) refers to target cellsco-incubated with effector cells without any TCB.

The results (FIGS. 39A and 39B) show target cell killing mediated byProtease activated MSLN TCB (8672) for NCI H596 and AsPC-1 cell lines.The protease activated TCBs nearly reaches the potency of MSLN TCB(8676) for NCI H596 and AsPC-1. The molecule containing thenon-cleavable GS linker (8673) does not induce killing in the indicatedconcentration range for both cell lines.

Example 21

Jurkat-NFAT Reporter Assay to Monitor Target Expression (FOLR1 TCB) andProtease Activity (Protease Activated FOLR1 TCB) in Primary TumorSamples

The intention of this assay was to show tumor target antigen (FolR1)expression and activity of tumor specific proteases like MMP9,Matriptase or Cathepsin in human tumor samples.

Jurkat-NFAT reporter cell line (Promega) is a human acute lymphaticleukemia reporter cell line with a NFAT promoter, expressing human CD3c.Luciferase expression can be measured, if the T cell bispecific moleculebinds the tumor target and the CD3c (crosslinkage). Luminescence ismeasured after addition of One-Glo substrate (Promega).

Primary tumor samples were received from Indivumed GmbH, Germany.Samples were shipped over night in transport medium. About 24 h aftersurgery the sample was cut in small pieces. 96-well white walled, flat(clear) bottom plate was prepared by adding 18 ul cold Matrigel(Matrigel (734-1101, Corning/VWR). Plate was incubated for 2 min at 37°C. before tumor pieces were added (triplicates). 33 ul of cold Matrigelwere added per well and plate was incubated again for 2 min at 37° C. 50ul of antibody dilution (in Jurkat medium without Hygromycine butcontaining 2×Penicillin/Streptomycine) was added per well and plate wasincubated for about 48 hours at 37° C., 5% CO₂.

Jurkat-NFAT reporter cells were harvested and viability was assessedusing ViCell. Cells were centrifuged at 350×g, 7 min before they wereresuspended in Jurkat medium without Hygromycine and 50 μl per well(50.000 cells/well) were added. Plate was incubated for 5 h at 37° C. ina humidified incubator before it was taken out for Luminescence readout. 80 ul of each well were transferred into a white walled 96-wellplate. 27 μl/well of ONE-Glo solution were added to each well andincubated for 10 min at room temperature in the dark. Luminescence wasdetected using WALLAC Victor3 ELISA reader (PerkinElmer2030), 1 sec/wellas detection time.

Jurkat NFAT reporter cells are activated after co-incubation with FolR1TCB (6298) and Protease activated FolR1 TCB containing MMP9-Matriptasecleavage site (8364). Protease activated FolR1 TCBs (8363, 8408) andcontrol TCBs (8409, 7235) do not induce Luciferase expression. Thedotted line indicates the baseline Luminescence for Jurkat NFAT cellsco-incubated with tumor (FIG. 40 ).

Example 22

Serum Stability of Protease Activated TCBs

Capillary electrophoresis of protease activated TCBs after incubation inhuman serum. Molecules were incubated for 0 or 14 days in human IgGdepleted serum at 37° C. in a humidified incubator (5% CO2). Allmolecules were purified by affinity chromatography (ProteinA) and thenanalyzed by Capillary electrophoresis.

100 ug of each molecule was added either in buffer (Histidine buffer(Bichsel) with 0.01% Tween-20) or in human serum (IgG depleted, SP1839,TL-15216, 16FSP63814). The concentration of the molecules was higherthan 2 mg/ml and the final concentration was 0.5 mg/ml. The pretreatmentfor one molecule (8408) was done with rhMatriptase (R&D Systems) for 24h at 37° C., 5% CO2 in a humidified incubator (otherwise pH of serumcould change). The samples for day 0 were directly frozen in liquidnitrogen and stored at −80° C. until analysis. Samples for day 14 wereincubated for 14 days at 37° C., 5% CO2 in a humidified incubator untilthey were also snap frozen.

Prior to CE-SDS analysis all samples were purified via HPLC affinitychromatography (Agilent technologies 1200series, column: Upchurchscientific C-130B, packaging material: Applied Biosystems POROS 20A 60μl, buffer: 10 mM Tris, 50 mM Glycine, 500 mM NaCl pH 8.0 and pH 2.0,injection volume: 100 μl, flow rate 1 ml/min, collection: peak based,neutralization: 0.5 M Na-phosphate pH 8.0 10% volume). Proteaseactivated TCB is stable in human IgG depleted serum for a minimum of 14days (FIGS. 41A-41C).

Example 23

Design of Anti Her2/Anti-CD3 and antiFolR1/Anti-CD3 T Cell Bispecific(TCB) Molecules with Anti CD3 scFv

Several T cell bispecific (TCB) molecules designed and are schematicallydepicted in FIGS. 42A-42F with their respective ID number. The followingmolecules were designed:

-   -   ID 8955: “Herceptarg 2+1 IgG, classic format, Herceptarg charged        variants, CD3 crossed (anti idiotypic scFv 4.32.63—MMP9—MK062        Matriptase—CD3—N-terminal fused to Herceptarg VH—inert Fc) with        N-terminal fused anti CD3 scFv 4.32.63 and MMP9—MK062        Matriptase” (FIG. 42A, SEQ ID NOs 81, 132, 133 and 136).    -   ID 8957: “Herceptarg 2+1 IgG, classic format, Herceptarg charged        variants, CD3 crossed (anti idiotypic scFv 4.32.63—non cleavable        GS linker—CD3—N-terminal fused to Herceptarg VH—inert Fc) with        N-terminal fused anti CD3 scFv 4.32.63 and non cleavable GS        linker” (FIG. 42B, SEQ ID NOs 81, 132, 133 and 135).    -   ID 8959: “Herceptarg 2+1 IgG, classic format, Herceptarg charged        variants, CD3 crossed (Herceptarg IgG with CD3—N-terminal fused        to Herceptarg VH—inert Fc)” (FIG. 42C, SEQ ID NOs 81, 132, 133        and 134).    -   ID 8997: “FolR1 36F2 2+1 IgG, classic format, FolR1 36F2 charged        variants, CD3 crossed (anti idiotypic scFv 4.32.63—MMP9—MK062        Matriptase—CD3—N-terminal fused to FolR1 36F2 VH—inert Fc) with        N-terminal fused anti CD3 scFv 4.32.63 and MMP9—MK062        Matriptase” (FIG. 42D, SEQ ID NOs 81, 137, 138 and 139).    -   ID 8998: “FolR1 36F2 2+1 IgG, classic format, FolR1 36F2 charged        variants, CD3 crossed (anti idiotypic scFv 4.32.63—non cleavable        GS linker—CD3—N-terminal fused to FolR1 36F2 VH—inert Fc) with        N-terminal fused anti CD3 scFv 4.32.63 and non cleavable GS        linker” (FIG. 42E, SEQ ID NOs 81, 137, 138 and 140).    -   ID 8996: “FolR1 36F2 2+1 IgG, classic format, FolR1 36F2 charged        variants, CD3 crossed (FolR1 36F2 IgG with CD3—N-terminal fused        to FolR1 36F2 VH—inert Fc)” (FIG. 42F, SEQ ID NOs 81, 137, 138        and 141).    -   The variable domains were subcloned in frame with the        pre-inserted domains into the respective recipient mammalian        expression vector. Protein expression is driven by an MPSV or        CMV (for Herceptarg) promoter and a synthetic polyA signal        sequence is present at the 3′ end of the CDS. In addition each        vector contains an EBV OriP sequence.

Example 24 Primary Cell Cytotoxicity Mediated by Protease ActivatedFolR1 TCB

T-cell killing mediated by protease activated FolR1 TCB molecule wasassessed on primary cell lines expressing low levels of FolR1 (FIG. 43). Human Peripheral blood mononuclear cells (PBMCs) were isolated frombuffy coats obtained from healthy human donors. Buffy coat was diluted1:1 with sterile PBS and layered over Histopaque gradient (Sigma,#H8889). After centrifugation (450×g, 30 minutes, w/o break, roomtemperature) the PBMC-containing interphase was transferred in a newfalcon tube that was subsequently filled with 50 ml of PBS. The mixturewas centrifuged (400× g, 10 minutes, room temperature), the supernatantwas discarded and the PBMC pellet was resuspended in 2 ml ACK buffer forErythrocytes lysis. After incubation for about 2-3 minutes at 37° C. thetubes were filled with sterile PBS to 50 ml and centrifuged for 10minutes at 350× g. This washing step was repeated once prior toresuspension of PBMCs in RPMI1640 medium containing 10% FCS, 1× GlutaMaxand 10% DMSO. PBMCs were slowly frozen in CoolCell® Cell FreezingContainers (BioCision) at −80° C. and then transferred to liquidnitrogen. One day before assay start adherent target cells wereharvested with Trypsin/EDTA, counted, checked for viability andresuspended in assay medium (RPMI1640, 2% FCS, 1× GlutaMax). Targetcells were plated at a density of 20 000 cells/well using 96-wellflat-bottom plates and incubated for about 20 h at 37° C. in ahumidified incubator. About 20 h before assay start PBMCs were thawed inRPMI1640 medium (10% FCS, 1× GlutaMax). PBMCs were centrifuged at 350 gfor 7 min. The pellet was resuspended in fresh medium (RPMI1640, 10%FCS, 1× GlutaMax) and incubated for max 24 h at 37° C. in a humidifiedincubator. On the day of the assay start PBMCs were harvested andcentrifuged at 350 g for 7 min. The pellet was resuspended in assaymedium and 0.2 mio PBMCs in 100 ul/well (E:T 10:1, based on the numberof seeded target cells) were added to the target cells. The moleculeswere diluted in assay medium (RPMI1640, 2% FCS, 1× GlutaMax) and 50ul/well were added at the indicated concentrations in triplicates beforethe plates were incubated for about 48 h, 72 h or 96 h at 37° C. in ahumidified incubator. Target cell killing was assessed after 48 h, 72 hand 96 h of incubation at 37° C., 5% CO2 by quantification of LDHrelease into cell supernatants by apoptotic/necrotic cells (LDHdetection kit, Roche Applied Science, #11 644 793 001). Maximal lysis ofthe target cells (=100%) was achieved by incubation of target cells with1% Triton X-100 20 h before LDH readout. Minimal lysis (=0%) refers totarget cells co-incubated with effector cells without any TCB.

Human Bronchial Epithelial Cell toxicity mediated by human PBMCs and 100nM or 10 nM of FolR1 TCB is higher compared to Protease activated TCB.

Example 25 FolR1 Negative Target Cell Cytotoxicity Mediated by ProteaseActivated FolR1 TCB

T-cell killing mediated by protease activated FolR1 TCB molecule wasassessed on FolR1 negative Mkn-45 cell line (FIG. 44 ). Human Peripheralblood mononuclear cells (PBMCs) were isolated from buffy coats obtainedfrom healthy human donors. Buffy coat was diluted 1:1 with sterile PBSand layered over Histopaque gradient (Sigma, #H8889). Aftercentrifugation (450×g, 30 minutes, w/o break, room temperature) thePBMC-containing interphase was transferred in a new falcon tube that wassubsequently filled with 50 ml of PBS. The mixture was centrifuged (400×g, 10 minutes, room temperature), the supernatant was discarded and thePBMC pellet was resuspended in 2 ml ACK buffer for Erythrocytes lysis.After incubation for about 2-3 minutes at 37° C. the tubes were filledwith sterile PBS to 50 ml and centrifuged for 10 minutes at 350× g. Thiswashing step was repeated once prior to resuspension of PBMCs inRPMI1640 medium containing 10% FCS, 1× GlutaMax and 10% DMSO. PBMCs wereslowly frozen in CoolCell® Cell Freezing Containers (BioCision) at −80°C. and then transferred to liquid nitrogen. One day before assay startadherent target cells were harvested with Trypsin/EDTA, counted, checkedfor viability and resuspended in assay medium (RPMI1640, 2% FCS, 1×GlutaMax). Target cells were plated at a density of 20 000 cells/wellusing 96-well flat-bottom plates and incubated for about 20 h at 37° C.in a humidified incubator. About 20 h before assay start PBMCs werethawed in RPMI1640 medium (10% FCS, 1× GlutaMax). PBMCs were centrifugedat 350 g for 7 min. The pellet was resuspended in fresh medium(RPMI1640, 10% FCS, 1× GlutaMax) and incubated for max 24 h at 37° C. ina humidified incubator. On the day of the assay start PBMCs wereharvested and centrifuged at 350 g for 7 min. The pellet was resuspendedin assay medium and 0.2 mio PBMCs in 100 ul/well (E:T 10:1, based on thenumber of seeded target cells) were added to the target cells. Themolecules were diluted in assay medium (RPMI1640, 2% FCS, 1× GlutaMax)and 50 ul/well were added at the indicated concentrations in triplicatesbefore the plates were incubated for about 48 h and 72 h at 37° C. in ahumidified incubator. Target cell killing was assessed after 48 h, 72 hand 96 h of incubation at 37° C., 5% CO2 by quantification of LDHrelease into cell supernatants by apoptotic/necrotic cells (LDHdetection kit, Roche Applied Science, #11 644 793 001). Maximal lysis ofthe target cells (=100%) was achieved by incubation of target cells with1% Triton X-100 20 h before LDH readout. Minimal lysis (=0%) refers totarget cells co-incubated with effector cells without any TCB. Proteaseactivated TCB did not induce target cell killing at 100 nM.

Examplary Sequences

SEQ  ID Construct No Amino acid Sequence LC Common lightQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK   1 chain pETR13197PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS anti CD3 (CH2527QIQLVQSGPELKKPGETVRISCKASGYTFTDYSIHWVKQAPG   2 VH_3-23(12) VL7-KCLKWMGWINTETGEPAYADDFKGRFAFSLETSASTAYLQI 46(13)) scFv15-NNLKNEDTATFFCAHPYDYDVLDYWGQGTSVTVSSGGGGS Matriptase MK062GGGGSGGGGSGGGGSDTVLTQSPASLGVSLGQRATISCRA CH2527 VH3_23-VH12SKSVSTSNYSYIHWYQQKPGQPPKLLIKYVSYLESGVPARFS CH1 FolR1 16D5 VHGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGCGTKL CH1 hum Fc knob PG EIKGGGGSGGGGSRQARVVNGGGGGSGGGGSGGGGS EV LALA, pETR15422QLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG (FIG. 45A)KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FolR1 16D5 VH CH1EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQA   3 Fc hole P329G LALAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTL HRYF, pETR15214YLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK anti CD3 (CH2527QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP   4 VH_3-23(12) VL7-GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS 46(13)) scFv 4.32.63LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG Matriptase MK062GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT CH2527 VH3_23-VH12CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS CH1 FolR1 16D5 VHGSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL CH1 hum Fc knob PG EIKGGGGSGGGGSRQARVVNGGGGGSGGGGSGGGGS EV LALA, pETR15599QLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG (FIG. 45B)KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti CD3 (CH2527QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP   5 VH_3-23(12) VL7-GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS 46(13)) scFv 4.32.63LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG non-cleavable linkerGGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT CH2527 VH3_23-VH12CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS CH1 FolR1 16D5 VHGSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL CH1 hum Fc knob PGEIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSEV LALA, pETR15603QLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG (FIG. 45C)KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti CD3 (CH2527QIQLVQSGPELKKPGETVRISCKASGYTFTDYSIHWVKQAPG   6 VH_3-23(12) VL7-KCLKWMGWINTETGEPAYADDFKGRFAFSLETSASTAYLQI 46(13)) scFv15 non-NNLKNEDTATFFCAHPYDYDVLDYWGQGTSVTVSSGGGGS cleavable linkerGGGGSGGGGSGGGGSDTVLTQSPASLGVSLGQRATISCRA CH2527 VH3_23-VH12SKSVSTSNYSYIHWYQ CH1 FolR1 16D5 VHQKPGQPPKLLIKYVSYLESGVPARFSGSGSGTDFTLNIHPVE CH1 hum Fc knob PGEEDAATYYCQHSREFPWTFGCGTKLEIKGGGGSGGGGSGG LALA, pETR14759GGSGGGGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGS (FIG. 45D)LRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKMK062 Protease linker GGGGSGGGGSRQARVVNGGGGGSGGGGSGGGGS   7Combined NF9/Mat5 GGGGSVHMPLGFLGPGRSRGSFPGGGGS   8 linker Combined MK062GGGGSGGGGSRQARVVNGGGGGSVPLSLYSGGGGGSGG   9 MMP9 GGS Combined MK062GGGGSGGGGSRQARVVNGVPLSLYSGGGGGSGGGGS  10 MMP9 H2527 CDR H1 Kabat TYAMN 11 CH2527 CDR H2 Kabat RIRSKYNNYATYYADSVKG  12 CH2527 CDR H3 KabatHGNFGNSYVSWFAY  13 FolR1 CDR H1 Kabat NAWMS  14 FolR1 CDR H2 KabatRIKSKTDGGTTDYAAPVKG  15 FolR1 CDR H3 Kabat PWEWSWYDY  16CLC CDR1 L1 Kabat GSSTGAVTTSNYAN  17 CLC CDR L2 Kabat GTNKRAP  18CLC CDR L3 Kabat ALWYSNLWV  19 Anti-ID 4.15.64 CDR DYSIH  20 H1 KabatAnti-ID 4.15.64 CDR WINTETGEPAYADDFKG  21 H2 Kabat Anti-ID 4.15.64 CDRPYDYDVLDY  22 H3 Kabat Anti-ID 4.15.64 CDR L1 RASKSVSTSNYSYIH  23 KabatAnti-ID 4.15.64 CDR L2 YVSYLES  24 Kabat Anti-ID 4.15.64 CDR L3QHSREFPWT  25 Kabat Anti-ID 4.32.63 CDR SYGVS  26 H1 KabatAnti-ID 4.32.63 CDR IIWGDGSTNYHSALIS  27 H2 Kabat Anti-ID 4.32.63 CDRGITTVVDDYYAMDY  28 H3 Kabat Anti-ID 4.32.63 CDR L1 RASENIDSYLA  29 KabatAnti-ID 4.32.63 CDR L2 AATFLAD  30 Kabat Anti-ID 4.32.63 CDR L3QHYYSTPYT  31 Kabat anti HER1 (GA201QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYKIHWVRQAP  32 heavy chain, pUC-Exp-GQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTSTAYM GA201-HC) (FIG. 45E)ELSSLRSEDTAVYYCARLSPGGYYVMDAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti HER1 (GA201 lightDIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQQKPG  33 chain, pUC-Exp-GA201-KAPKRLIYNTNNLQTGVPSRFSGSGSGTEFTLTISSLQPEDF LC) (FIG. 45F)ATYYCLQHNSFPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC anti HER1 (anti-GA201EVQLEQSGPVLVKPGTSVKMSCKASGYTFTDYYINWIIQSHG  34 VH-VL scFv MMP K CLEWIGVINPDSGGTDYNQNFKGKATLTVDKSSTTAYMELT cleavable linker G4SSLTSEDSAVYYCARRDSYGFDYWGQGTTLTVSSGGGGSGG GA201 light chain, pUC-GGSGGGGSGGGGSDIVLTQTPKFLLVPAGDRITMTCKASLS I_GA201_MMP_LC)VTNDVAWYQQKPGQSPKLLLYYASNRNAGVPDRFTGSGYG (FIG. 45G)TDFTFTITTLQAEDLAVYFCQQDYTSPPTFG C GTKLEIRGGGGSGGGGSGPLGLWSQGGGGSGGGGSGGGGSGGDIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQQKPGKAPKRLIYNTNNLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSFPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MMP Protease linkerGGGGSGGGGSGPLGLWSQGGGGSGGGGSGGGGSGG  35 Protease recognition RQARVVNG 36 site 1 Protease recognition VHMPLGFLGPGRSRGSFP  37 site 2Protease recognition RQARVVNGXXXXXVPLSLYSG  38 site 3Protease recognition RQARVVNGVPLSLYSG  39 site 4 Protease recognitionPLGLWSQ  40 site 5 4.15.64 Anti-idiotypicQIQLVQSGPELKKPGETVRISCKASGYTFTDYSIHWVKQAPG  41 scFvKCLKWMGWINTETGEPAYADDFKGRFAFSLETSASTAYLQINNLKNEDTATFFCAHPYDYDVLDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDTVLTQSPASLGVSLGQRATISCRASKSVSTSNYSYIHWYQQKPGQPPKLLIKYVSYLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGCGTKL EIK 4.32.63 Anti-idiotypicQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP  42 scFvGKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNSLQTDDTATYYCAKGITTWDDYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFSGSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL EIK Anti-CD3 variable heavyEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQA  43 chain (VH)PGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVT VSS CD3 heavy chain TYAMN  44(VH)_CDR1 CD3 heavy chain RIRSKYNNYATYYADSVKG  45 (VH)_CDR2CD3 heavy chain HGNFGNSYVSWFAY  46 (VH)_CDR3 Anti-FolR1 16D5EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQA  47 variable regionPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSS anti-idiotypic GA201 DYYIN  48CDR H1 Kabat anti-idiotypic GA201 VINPDSGGTDYNQNFKG  49 CDR H2 Kabatanti-idiotypic GA201 RDSYGFDY  50 CDR H3 Kabat anti-idiotypic GA201KASLSVTNDVA  51 CDR L1 Kabat anti-idiotypic GA201 YASNRNA  52CDR L2 Kabat anti-idiotypic GA201 QQDYTSPPT  53 CDR L3 Kabat hu CD3EMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSI  54SGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQR RI LC Common lightQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK  55 chain pETR13197PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP V regionEDEAEYYCALWYSNLWVFGGGTKLTVL GA201 CDR H1 Kabat DYKIH  56GA201 CDR H2 Kabat YFNPNSGYSTYAQKFQG  57 GA201 CDR H3 Kabat LSPGGYYVMDA 58 GA201 CDR L1 Kabat RASQGINNYLN  59 GA201 CDR L2 Kabat NTNNLQT  60GA201 CDR L3 Kabat LQHNSFPT  61 DNA Sequence LC CommonCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGC  62 light chainGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACC pETR13197ACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGG CCCCCACCGAGTGCAGCTGAanti CD3 CAGATCCAGCTGGTGCAGAGCGGCCCTGAGCTGAAGAAACCCGGC  63 (CH2527GAGACAGTGCGGATCAGCTGCAAGGCCAGCGGCTACACCTTCACC VH_3-23(12)GACTACAGCATCCACTGGGTCAAGCAGGCCCCTGGCAAGTGCCTG VL7-46(13))AAGTGGATGGGCTGGATCAACACCGAGACAGGCGAGCCCGCCTAC scFv15-GCCGACGATTTCAAGGGCAGATTCGCCTTCAGCCTGGAAACCAGC MatriptaseGCCAGCACCGCCTACCTGCAGATCAACAACCTGAAGAACGAGGAC MK062ACCGCCACCTTTTTCTGCGCCCACCCCTACGACTACGACGTGCTG CH2527GATTATTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGCGGAGGC VH3_23-VH12GGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGG CH1 FolR1GAGGCGGATCTGATACCGTGCTGACACAGAGCCCTGCCAGCCTGG 16D5 VH CH1GAGTGTCCCTGGGACAGAGAGCCACCATCAGCTGTCGGGCCAGCA hum Fc knobAGAGCGTGTCCACCAGCAACTACAGCTATATCCACTGGTATCAGCA PG LALA,GAAGCCCGGCCAGCCCCCCAAGCTGCTGATCAAATACGTGTCCTA pETR15422CCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGGCAGCGG (FIG. 45H)CACCGACTTCACCCTGAACATCCACCCCGTGGAAGAGGAAGATGCCGCCACCTACTACTGCCAGCACAGCAGAGAGTTCCCTTGGACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTAGACAGGCCAGAGTCGTGAACGGGGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA FolR1 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGC  64 VH CH1 FcGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCC hole P329GAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCT LALA HRYF,CGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCAC pETR15214GGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti CD3CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC  65 (CH2527CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG MatriptaseAGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC MK062GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC CH2527TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG VH3_23-VH12TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG CH1 FolR1GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC 16D5 VH CH1CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT hum Fc knobGCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC PG LALA,AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT pETR15599TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG (FIG. 45I)GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTAGACAGGCCAGAGTCGTGAACGGGGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGGGGCGGAGGATCCGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGGTTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti CD3CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC  66 (CH2527CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG non-AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC cleavableGCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC linkerTACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG CH2527TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG VH3_23-VH12GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH1 FolR1CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT 16D5 VH CH1GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC hum Fc knobAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT PG LALA,TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG pETR15603GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG (FIG. 45J)TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGCTCCGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT GA anti CD3CAGATCCAGCTGGTGCAGAGCGGCCCTGAGCTGAAGAAACCCGGC  67 (CH2527GAGACAGTGCGGATCAGCTGCAAGGCCAGCGGCTACACCTTCACC VH_3-23(12)GACTACAGCATCCACTGGGTCAAGCAGGCCCCTGGCAAGTGCCTG VL7-46(13))AAGTGGATGGGCTGGATCAACACCGAGACAGGCGAGCCCGCCTAC scFv15 non-GCCGACGATTTCAAGGGCAGATTCGCCTTCAGCCTGGAAACCAGC cleavableGCCAGCACCGCCTACCTGCAGATCAACAACCTGAAGAACGAGGAC linkerACCGCCACCTTTTTCTGCGCCCACCCCTACGACTACGACGTGCTG CH2527GATTATTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGCGGAGGC VH3_23-VH12GGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGG CH1 FolR1GAGGCGGATCTGATACCGTGCTGACACAGAGCCCTGCCAGCCTGG 16D5 VH CH1GAGTGTCCCTGGGACAGAGAGCCACCATCAGCTGTCGGGCCAGCA hum Fc knobAGAGCGTGTCCACCAGCAACTACAGCTATATCCACTGGTATCAGCA PG LALA,GAAGCCCGGCCAGCCCCCCAAGCTGCTGATCAAATACGTGTCCTA pETR14759CCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGGCAGCGG (FIG. 45K)CACCGACTTCACCCTGAACATCCACCCCGTGGAAGAGGAAGATGCCGCCACCTACTACTGCCAGCACAGCAGAGAGTTCCCTTGGACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGCTCCGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA MK062GGCGGGGGAGGCTCCGGAGGCGGCGGAAGTAGACAGGCCAGAG  68 ProteaseTCGTGAACGGGGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGG linker GGGCGGAGGATCCanti HER1 CAGGTGCAGCTGGTCCAGAGCGGCGCCGAGGTGAAGAAACCCGG  69 (GA201 heavyGTCCTCTGTCAAGGTGTCATGCAAGGCTAGCGGATTCACCTTTACA chain, pUC-GACTACAAAATCCACTGGGTTAGGCAGGCACCTGGCCAAGGACTC Exp-GA201-GAATGGATGGGGTATTTCAACCCAAATTCCGGCTACTCTACCTATG HC)CCCAGAAGTTTCAGGGAAGAGTGACTATTACAGCTGATAAGAGTACCAGCACTGCATACATGGAGCTGTCCTCTCTTCGCTCAGAGGACACCGCCGTCTACTATTGTGCTCGGCTGAGCCCCGGTGGCTACTATGTGATGGATGCATGGGGGCAGGGAACAACCGTAACAGTGTCCTCTGCGTCGACTAAGGGCCCTTCAGTTTTTCCACTCGCCCCCAGTAGCAAGTCCACATCTGGGGGTACCGCTGCCCTGGGCTGCCTTGTGAAAGACTATTTCCCTGAACCAGTCACTGTGTCATGGAATAGCGGAGCCCTGACCTCCGGTGTACACACATTCCCCGCTGTGTTGCAGTCTAGTGGCCTGTACAGCCTCTCCTCTGTTGTGACCGTCCCTTCAAGCTCCCTGGGGACACAGACCTATATCTGTAACGTGAATCATAAGCCATCTAACACTAAAGTAGATAAAAAAGTGGAGCCCAAGAGTTGCGACAAAACACACACCTGTCCCCCTTGCCCAGCCCCCGAGCTTCTGGGAGGCCCTAGCGTCTTTCTCTTCCCACCCAAGCCTAAGGATACTCTGATGATATCCAGGACCCCAGAAGTTACATGCGTGGTCGTGGACGTCTCACACGAGGACCCCGAAGTGAAATTTAACTGGTACGTTGATGGTGTGGAAGTCCATAATGCCAAGACCAAGCCTAGAGAGGAGCAATACAACAGTACATATCGCGTGGTAAGCGTGTTGACCGTTCTCCACCAGGACTGGCTCAATGGGAAAGAATACAAGTGTAAAGTGTCCAACAAAGCTCTGCCAGCACCCATCGAGAAGACTATTTCTAAGGCCAAAGGCCAGCCCCGGGAGCCTCAGGTCTATACACTTCCACCCTCAAGGGATGAACTGACCAAGAACCAAGTGAGCTTGACTTGCCTGGTAAAGGGGTTCTACCCTTCCGACATCGCTGTGGAGTGGGAGTCTAATGGACAACCAGAAAACAATTACAAAACCACACCCCCTGTCCTCGACAGTGATGGCAGCTTTTTCCTGTATAGCAAACTTACCGTTGACAAGTCCAGATGGCAGCAGGGAAACGTGTTCTCATGTAGCGTCATGCACGAAGCTTTGCATAACCACTACACACAGAAAAGCCTCAGCCTGAGTCCAGGGAAG anti HER1GACATCCAAATGACCCAGTCACCTAGTAGCCTCTCCGCCTCTGTTG  70 (GA201 lightGCGACAGGGTGACAATTACATGCAGAGCTTCACAGGGTATCAACAA chain, pUC-TTACCTGAACTGGTATCAGCAGAAACCAGGGAAGGCCCCCAAGCG Exp-GA201-CTTGATATATAACACCAATAACCTGCAAACTGGCGTCCCTAGCCGG LC)TTCTCCGGATCTGGTAGTGGCACCGAATTTACACTCACCATCAGCTCCCTGCAGCCAGAGGATTTCGCCACATACTATTGTCTTCAGCATAATTCTTTCCCCACCTTTGGGCAAGGAACTAAACTGGAGATTAAGCGTACTGTCGCCGCTCCCTCTGTGTTCATTTTTCCTCCAAGTGATGAGCAGCTCAAAAGCGGTACCGCATCCGTTGTGTGCCTGCTTAACAACTTCTATCCCCGGGAAGCCAAGGTCCAATGGAAGGTGGACAATGCTCTGCAGTCAGGAAACAGTCAGGAGAGCGTAACCGAGCAGGATTCCAAAGACTCTACTTACTCATTGAGCTCCACCCTGACACTCTCTAAGGCAGACTATGAAAAGCATAAAGTGTACGCCTGTGAGGTTACCCACCAGGGCCTGAGTAGCCCTGTGACAAAGTCCTTCAATAGGGGAGAGTGC HER1 (anti-GAGGTTCAGCTGGAGCAGTCAGGACCTGTGCTGGTGAAGCCTGGG  71 GA201 VH-ACTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTG VL scFv MMPACTACTATATAAACTGGATAATACAGAGCCATGGAAAGTGTCTTGAG cleavableTGGATTGGAGTTATTAATCCTGACAGCGGTGGTACTGACTACAACC linker G4SAGAACTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAC GA201 lightCACAGCCTACATGGAACTCACTAGCCTGACATCTGAGGACTCTGCA chain, pUC-GTCTATTATTGTGCAAGAAGGGATTCTTACGGCTTTGACTACTGGG I_GA201_MMGCCAAGGCACCACTCTCACAGTCTCCTCAGGCGGAGGTGGCTCAG P_LC)GGGGAGGCGGTAGCGGCGGAGGTGGCTCAGGGGGAGGCGGTAGCGACATTGTGCTGACCCAGACTCCCAAATTCCTGCTTGTGCCAGCAGGAGACAGGATTACCATGACCTGCAAGGCCAGTCTGAGTGTGACTAATGATGTAGCTTGGTATCAACAGAAACCAGGGCAGTCTCCTAAACTGCTGTTATACTATGCATCCAATCGCAACGCTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCACCACTTTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATACCTCTCCTCCGACGTTCGGTTGTGGCACCAAGCTAGAAATCCGTGGTGGCGGCGGTTCTGGCGGAGGGGGTTCTGGCCCCCTGGGGCTATGGAGCCAGGGTGGCGGCGGTTCTGGCGGAGGGGGTTCTGGCGGTGGTGGCTCTGGCGGTGACATCCAAATGACCCAGTCACCTAGTAGCCTCTCCGCCTCTGTTGGCGACAGGGTGACAATTACATGCAGAGCTTCACAGGGTATCAACAATTACCTGAACTGGTATCAGCAGAAACCAGGGAAGGCCCCCAAGCGCTTGATATATAACACCAATAACCTGCAAACTGGCGTCCCTAGCCGGTTCTCCGGATCTGGTAGTGGCACCGAATTTACACTCACCATCAGCTCCCTGCAGCCAGAGGATTTCGCCACATACTATTGTCTTCAGCATAATTCTTTCCCCACCTTTGGGCAAGGAACTAAACTGGAGATTAAGCGTACTGTCGCCGCTCCCTCTGTGTTCATTTTTCCTCCAAGTGATGAGCAGCTCAAAAGCGGTACCGCATCCGTTGTGTGCCTGCTTAACAACTTCTATCCCCGGGAAGCCAAGGTCCAATGGAAGGTGGACAATGCTCTGCAGTCAGGAAACAGTCAGGAGAGCGTAACCGAGCAGGATTCCAAAGACTCTACTTACTCATTGAGCTCCACCCTGACACTCTCTAAGGCAGACTATGAAAAGCATAAAGTGTACGCCTGTGAGGTTACCCACCAGGGCCTGAGTAGCCCTGTGACAAAGTCCT TCAATAGGGGAGAGTGCAmino acid Sequence anti CD3 (CH2527QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP  72 VH_3-23(12) VL7-GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS 46(13)) scFv 4.32.63LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG MMP9 MatriptaseGGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT MK062 CH2527CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS VH3_23-VH12 CH1GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL FolR1 16D5 VH CH1EIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSEVQ hum Fc knob PG LALA,LLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGK pETR16546 (FIG. 45L)GLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FolR1 16D5 HCEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQA  73 CH2527-VH3_23-12PGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTL HC Fc knob PG LALA,YLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSA pCON999 (FIG. 45M)STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti ID CD3 scFvQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP  74 4.32.63 MK062GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS protease site CD3 VLLQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG CLambda, pETR16544GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFSGSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKLEIKGGGGSGGGGSRQARVVNGGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS anti ID CD3 scFvQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP  75 4.32.63 non-cleavableGKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS linker CD3 VLLQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG CLambda, pETR16545GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFSGSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS aMSLN RG7787 VHQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQA  76 CH1 EE CD3 CH2527-PGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVY VH3_23-12 VLCH1 FcMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSSA knob PG LALA,STKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWN pETR15445SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKaMSLN RG7787 VH QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQA  77CH1EE Fc hole P329G PGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYLALA, pETR15444 MELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aMSLN RG7787 VL CkDIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGK  78 RK, pETR15443APKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC anti ID CH2527 4.32.63QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP  79 CD3 CH2527 VH 23-12GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS Ck, MMP9-MK062 site,LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG pETR16758GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFSGSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKLEIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGECanti ID CH2527 4.32.63 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP  80CD3 CH2527 VH 23-12 GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNSCk, non-cleavable LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGGlinker, pETR16759 GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFSGSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGECCD3 CH2527 VH 23- EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQA  8112-Ck, pETR13811 PGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGECanti CD3 (CH2527 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP  82VH_3-23(12) VL7- GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS46(13)) scFv 4.32.63 LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGGMMP9 Matriptase GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT MK062 aMSLN VHCRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS CH1 EE CH2527-GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL VL7_46-13 CH1 hum FcEIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSQAV knob PG LALA,VTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPG pETR16751QAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGKanti CD3 (CH2527 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP  83VH_3-23(12) VL7- GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS46(13)) scFv 4.32.63 LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGGnon-cleavable linker GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITaMSLN VH CH1 EE CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFSCH2527-VL7_46-13 GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKLCH1 hum Fc knob PG EIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSQ LALA, pETR16752AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKCH2527 XFab aMSLN QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK  84RG7787 HC EE Fc PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP knob PG LALA,EDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLA pETR16764PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKanti CD3 (CH2527 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP  85VH_3-23(12) VL7- GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS46(13)) scFv 4.32.63 LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGGCathepsin S/B site GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITCH2527 VH3_23-VH12 CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFSCH1 FolR1 16D5 VH GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKLCH1 hum Fc knob PG EIKGGGGSGGGGSGGGGSFVGGTGGGGSGGGGSGGSEVLALA, pETR16550 QLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Combined MMP9GGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGS  86 MK062, 33 AA for CD3 Combined MMP9GGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSGG  87 MK062, 35 AA for Her1Cathepsin S/B GGGGSGGGGSGGGGSFVGGTGGGGSGGGGSGGS  88 KKAAPVNGGGGGSGGGGSKKAAPVNGGGGGSGGGGSGGGGS  89 PMAKKVNGGGGGSGGGGSPMAKKVNGGGGGSGGGGSGGGGS  90 QARAKVNGGGGGSGGGGSQARAKVNGGGGGSGGGGSGGGGS  91 MMP9GGGGSGGGGSVHMPLGFLGPGGGGSGGGGSGGS  92 QARAKGGGGSGGGGSQARAKGGGGSGGGGSGGGGSGGS  93 MMP9-PMAKKGGGGSVHMPLGFLGPPMAKKGGGGSGGGGSGGS  94 KKAAPGGGGSGGGGSKKAAPGGGGSGGGGSGGGGSGGS  95 PMAKKGGGGSGGGGSPMAKKGGGGSGGGGSGGGGSGGS  96 Protease recognitionVHMPLGFLGPRQARVVNG  97 site 6 Protease recognition FVGGTG  98 site 7Protease recognition KKAAPVNG  99 site 8 Protease recognition PMAKKVNG100 site 9 Protease recognition QARAKVNG 101 site 10Protease recognition VHMPLGFLGP 102 site 11 Protease recognition QARAK103 site 12 Protease recognition VHMPLGFLGPPMAKK 104 site 13Protease recognition KKAAP 105 site 14 Protease recognition PMAKK 106site 15 aMSLN CDR H1 Kabat GYTMN 107 aMSLN CDR H2 KabatLITPYNGASSYNQKFRG 108 aMSLN CDR H3 Kabat GGYDGRGFDY 109aMSLN CDR L1 Kabat SASSSVSYMH 110 aMSLN CDR L2 Kabat DTSKLAS 111aMSLN CDR L3 Kabat QQWSKHPLT 112 aMSLN VHQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQA 113PGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS aMSLN VLDIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGK 114APKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQWSKHPLTFGQGTKLEIKaHER1 VH QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYKIHWVRQAP 115GQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQGTTVTVSS aHER1 VLDIQMTQSPSSLSASVGDRVTiTCRASQGSNNYLNWYQQKPGKA 116PKRLIYNTNNLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQHNSFPTFGQGTKLEIKDNA Sequence LC Common CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGC 117light chain GGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACC pETR13197ACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGG CCCCCACCGAGTGCAGCTGAanti CD3 CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 118 (CH2527CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG MMP9AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC MatriptaseGCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC MK062TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG CH2527TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG VH3_23-VH12GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH1 FolR1CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT 16D5 VH CH1GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC hum Fc knobAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT PG LALA,TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG pETR16546GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG (FIG. 45N)TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTGTGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCGTGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGGGGCGGAGGATCCGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGGTTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA FolR1 16D5GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGC 119 HC CH2527-GGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCC VH3_23-12AACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCT HC Fc knobCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCAC PG LALA,GGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGAC pCON999GATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti ID CD3CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 120 scFv 4.32.63CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC MK062AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG protease siteGAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC CD3 VLAGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG CLambda,AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC pETR16544GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGACTACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCCCTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACATGCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGCAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCTTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCGGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTAGACAGGCCAGAGTCGTGAACGGGGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGCGGAGGATCCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCC CCCACCGAGTGCAGCTGAanti ID CD3 CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 121scFv 4.32.63 CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC non-cleavableAGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG linker CD3 VLGAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC CLambda,AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG pETR16545AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACCGCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGACTACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCCCTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACATGCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGCAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCTTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCGGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCC CCCACCGAGTGCAGCTGA aMSLNCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGC 122 RG7787 VHGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACC CH1 EE CD3GGCTACACCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTG CH2527-GAATGGATGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTAC VH3_23-12AACCAGAAGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGC VLCH1 FcACCTCCACCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGAC knob PGACCGCCGTGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGC LALA,TTCGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTA pETR15445GCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACTGCCGCTCTGGGCTGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCTCTGACCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACGAGAAGGTGGAACCCAAGTCCTGCGACGGTGGCGGAGGTTCCGGAGGCGGAGGATCCCAGGCTGTCGTGACCCAGGAACCCTCCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACCTGTGGATCTTCTACCGGCGCTGTGACCACCTCCAACTACGCCAATTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCTCCGGTTCTCTGCTGGGCGGCAAGGCTGCCCTGACTCTGTCTGGTGCTCAGCCTGAGGACGAGGCCGAGTACTACTGCGCCCTGTGGTACTCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGTCCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATAAGACCCACACCTGTCCCCCCTGCCCTGCTCCTGAAGCTGCTGGTGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGCTCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA aMSLNCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGC 123 RG7787 VHGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACC CH1EE FcGGCTACACCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTG hole P329GGAATGGATGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTAC LALA,AACCAGAAGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGC pETR15444ACCTCCACCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGCTTCGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA aMSLNGACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTG 124 RG7787 VLGGCGACAGAGTGACCATCACCTGTAGCGCCAGCAGCAGCGTGTCC Ck RK,TACATGCACTGGTATCAGCAGAAGTCCGGCAAGGCCCCCAAGCTG pETR15443CTGATCTACGACACCAGCAAGCTGGCCTCCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCTCCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTGGTCCAAGCACCCCCTGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATCGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGTTAG anti IDCAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 125 CH2527CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC 4.32.63 CD3AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG CH2527 VHGAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC 23-12 Ck,AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG MMP9-MK062AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC site,GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC pETR16758TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCCCTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACATGCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGCAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCTTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCGGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTGTGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCGTGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTT TCAACCGGGGCGAGTGCTGAanti ID CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 126 CH2527CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC 4.32.63 CD3AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG CH2527 VHGAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC 23-12 Ck,AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG non-cleavableAGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC linker,GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC pETR16759TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCCCTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACATGCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGCAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCTTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCGGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTC TTTCAACCGGGGCGAGTGCTGACD3 CH2527 GAAGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGG 127 VH 23-12-CGGATCTCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCC Ck,ACCTACGCCATGAACTGGGTGCGACAGGCTCCTGGCAAGGGCCTG pETR13811GAATGGGTGTCCCGGATCAGATCCAAGTACAACAACTACGCCACCTACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCTCGGGACGACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACTCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCT TTCAACCGGGGCGAGTGCTGAanti CD3 CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 128 (CH2527CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG MMP9AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC MatriptaseGCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC MK062TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG aMSLN VHTCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG CH1 EEGGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH2527-CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT VL7_46-13GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC CH1 hum FcAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT knob PGTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG LALA,GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG pETR16751TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTGTGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCGTGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACACCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAATGGATGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTACAACCAGAAGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGCACCTCCACCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGCTTCGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti CD3CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 129 (CH2527CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG non-cleavableAGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC linker aMSLNGCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC VH CH1 EETACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG CH2527-TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG VL7_46-13GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH1 hum FcCTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT knob PGGCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC LALA,AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT pETR16752TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCGGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACACCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAATGGATGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTACAACCAGAAGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGCACCTCCACCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGCTTCGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA CH2527 XFabCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGC 130 aMSLNGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACC RG7787 HCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCC EE Fc knobTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACC PG LALA,CCTGCCAGATTCTCCGGTTCTCTGCTGGGCGGCAAGGCTGCCCTG pETR16764ACTCTGTCTGGTGCTCAGCCTGAGGACGAGGCCGAGTACTACTGCGCCCTGTGGTACTCCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGTCCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACACCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAATGGATGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTACAACCAGAAGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGCACCTCCACCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGCTTCGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti CD3CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 131 (CH2527CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG Cathepsin S/BAGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC site CH2527GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC VH3_23-VH12TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG CH1 FolR1TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG 16D5 VH CH1GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC hum Fc knobCTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT PG LALA,GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC pETR16550AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCTTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCGGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTGGAGGCGGCGGAAGTTTCGTGGGGGGGACCGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGGGGGATCCGAGGTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGGAGGAGGGTCCGGGGGCGGAGGATCCGAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGGTTCACCTTCTCCAACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Amino acid SequencepETR16859 Omnitarg EVQLVESGGGLVQPGGSLRLSCAASGFTFNDYTMDWVRQA 132aff.mat variant Fab cv- PGKGLEWVADVNPNSGGSIVNRRFKGRFTLSVDRSKNTLYLFc hole PG LALA QMNSLRAEDTAVYYCARNLGPFFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK pETR16860 HerceptargDIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPG 133 common CLkRKKAPKLLIYSASFRYTGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC pETR17605 CD3X FabQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 134 Herceptin HC chargedPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP variants Fc knob PGEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLA LALAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKpETR17606 anti CD3 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 135(CH2527 VH_3-23(12) GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNSVL7-46(13)) scFv LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG4.32.63 non cleavable GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITlinker aHerceptin VH CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFSCH1 EE CH2527- GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKLVL7_46-13 CH1 hum Fc EIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSQ knob PG LALAAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKpETR17607 anti CD3 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 136(CH2527 VH_3-23(12) GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNSVL7-46(13)) scFv LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG 4.32.63 MMP9GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT Matriptase MK062CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS aHerceptin VH CH1 EEGSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL CH2527-VL7_46-13EIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSQAV CH1 hum Fc knob PGVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPG LALAQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGKFolR1 36F2 VH CH1 EE QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQA 137Fc hole PG LALA PGQGLEWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVY pETR14797MELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FolR1 36F2 VLCk RK,EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP 138 pETR14798GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYTNEHYYTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC anti CD3 (CH2527QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 139 VH_3-23(12) VL7-GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS 46(13)) scFv 4.32.63LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG MMP9 MatriptaseGGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT MK062 aFolR1 36F2CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS VH CH1 EE CH2527-GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL VL7_46-13 CH1 hum FcEIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSQAV knob PG LALAVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPG pETR17621QAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGKanti CD3 (CH2527 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 140VH_3-23(12) VL7- GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS46(13)) scFv 4.32.63 LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGGnon cleavable linker GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTITaFolR1 36F2 VH CH1 CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFSEE CH2527-VL7_46-13 GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKLCH1 hum Fc knob PG EIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSQ LALA pETR17622AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKFolR1 36F2 classic QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 141format: CH2527 XFab PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP36F2 HC EE Fc knob EDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPG LALA pETR17623 PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKHerceptin/Omnitarg DYTMD 142 CDR H1 Kabat Herceptin/OmnitargDVNPNSGGSIVNRRFKG 143 CDR H2 Kabat Herceptin/Omnitarg NLGPFFYFDY 144CDR H3 Kabat Perjeta CDR H1 Kabat TSNYANW 145 Perjeta CDR H2 KabatGTNKRAPGTPARFSGSLLGG 146 Perjeta CDR H3 Kabat TKLTV 147 CLC CDR L1 KabatKASQDVSTAVA 148 CLC CDR L2 Kabat SASFRYT 149 CLC CDR L3 Kabat QQHYTTPPT150 36F2 CDR H1 Kabat SYYMH 151 36F2 CDR H2 Kabat IINPSGGSTSYAQKFQG 15236F2 CDR H3 Kabat SFFTGFHLDY 153 36F2 CDR L1 Kabat RASQSVSSSYLA 15436F2 CDR L2 Kabat GASSRAT 155 36F2 CDR L3 Kabat QQYTNEHYYT 156Anti-FolR1 36F2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQA 157variable region VH PGQGLEWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVYMELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSS Anti-FolR1 36F2EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP 158 variable region VLGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQYTNEHYYTFGQGTKVEIKHerceptarg variable EVQLVESGGGLVQPGGSLRLSCAASGFTFNDYTMDWVRQA 159region VH1 PGKGLEWVADVNPNSGGSIVNRRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPFFYFDYWGQGTLVTVSS Herceptarg variableEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP 160 region VH2GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS Herceptarg commonDIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPG 161 variable region VLKAPKLLIYSASFRYTGVPSRFSGSRSGTDFTLTISSLQPEDFA TYYCQQHYTTPPTFGQGTKVEIKpETR16859 DNA Sequence OmnitargGAAGTTCAGCTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGT 162 aff.mat variantGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTAACG Fab cv-FcATTATACCATGGATTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGA hole PG LALAATGGGTTGCAGATGTTAATCCGAATAGCGGTGGTAGCATTGTTAACCGTCGTTTTAAAGGTCGTTTTACCCTGAGCGTTGATCGTAGCAAAAATACCCTGTATCTGCAAATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTATTATTGTGCACGTAACCTGGGTCCGTTCTTCTACTTTGATTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA pETR16860GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTG 163 HerceptargGGCGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCC commonACAGCCGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAG CLkRKCTGCTGATCTACAGCGCCAGCTTCCGGTACACCGGCGTGCCCAGCAGATTCAGCGGCAGCAGATCCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCCCCCACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATCGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTAG pETR17606CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 164 anti CD3CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC (CH2527AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VH_3-23(12)GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC VL7-46(13))AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG scFv 4.32.63AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC non cleavableGCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC linkerTACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG aHerceptin VHTCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG CH1 EEGGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH2527-CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT VL7_46-13GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC CH1 hum FcAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT knob PGTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG LALAGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCGAGGTCCAGCTGGTCGAGTCTGGAGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGACTGAGCTGCGCCGCCAGCGGATTCAACATCAAGGACACCTACATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGACTGGAGTGGGTGGCCAGAATCTACCCCACCAACGGCTACACAAGATACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCTCTAGATGGGGAGGCGAGGGCTTCTACGCCATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA pETR17607CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 165 anti CD3CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC (CH2527AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VH_3-23(12)GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC VL7-46(13))AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG scFv 4.32.63AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC MMP9GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC MatriptaseTACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG MK062TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG aHerceptin VHGGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH1 EECTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT CH2527-GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC VL7_46-13AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT CH1 hum FcTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG knob PGGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG LALATGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTGTGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCGTGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCGAGGTCCAGCTGGTCGAGTCTGGAGGAGGACTGGTGCAGCCAGGCGGATCTCTGAGACTGAGCTGCGCCGCCAGCGGATTCAACATCAAGGACACCTACATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGACTGGAGTGGGTGGCCAGAATCTACCCCACCAACGGCTACACAAGATACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACAGCCGTGTACTACTGCTCTAGATGGGGAGGCGAGGGCTTCTACGCCATGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA FolR1 36F2CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 166 VH CH1 EECTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC Fc hole PGCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGA LALAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGC pETR14797GCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA FolR1 36F2GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 167 VLCk RK,GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCA pETR14798GCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATACCAACGAACATTATTATACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATCGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGTTAG anti CD3CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 168 (CH2527CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG MMP9AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC MatriptaseGCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC MK062TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG aFolR1 36F2TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG VH CH1 EEGGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH2527-CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT VL7_46-13GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC CH1 hum FcAGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT knob PGTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG LALAGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG pETR17621TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTGTGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCGTGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (CH2527AGAGTCCGGCCCTGGACTGGTGGCCCCTAGCCAGAGCCTGAGCAT 169 VH_3-23(12)CACCTGTACCGTGTCCGGCTTCAGCCTGACCAGCTACGGCGTGTC VL7-46(13))ATGGGTGCGCCAGCCTCCAGGCAAGTGTCTGGAATGGCTGGGCAT scFv 4.32.63CATCTGGGGCGACGGCAGCACCAATTACCACAGCGCCCTGATCAG non cleavableCAGACTGAGCATCTCCAAGGACAACAGCAAGAGCCAGGTGTTCCT linker aFolR1GAAGCTGAACAGCCTGCAGACCGACGACACCGCCACCTACTACTG 36F2 VH CH1CGCCAAGGGCATCACCACCGTGGTGGACGACTACTACGCTATGGA EE CH2527-CTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGCGGAGGCG VL7_46-13GAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGGG CH1 hum FcAGGCGGAAGCGATATCCAGATGACCCAGAGCCCTGCCAGCCTGTC knob PGTGCCTCTGTGGGCGAGACAGTGACCATCACATGCCGGGCCAGCGA LALAGAACATCGACAGCTACCTGGCCTGGTATCAGCAGAAGCAGGGCAA pETR17622GAGCCCCCAGCTGCTGGTGTACGCCGCCACCTTTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCGGCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATGGCGGAGGAGGGTCTGGAGGCGGAGGGTCCCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAATGAFolR1 36F2 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGC 170classic format: GGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCCH2527 XFab ACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCC 36F2 HC EETTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACC Fc knob PGCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTG LALAACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGC pETR17623GCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATGGCGGAGGAGGGTCTGGAGGCGGAGGGTCCCAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A protease-activatable T cell activating bispecific moleculecomprising (a) a first antigen binding moiety capable of specificbinding to CD3; (b) a second antigen binding moiety capable of specificbinding to a target cell antigen; and (c) a masking moiety covalentlyattached to the T cell bispecific binding molecule through aprotease-cleavable linker, wherein the masking moiety is capable ofspecific binding to the idiotype of the first or the second antigenbinding moiety thereby reversibly concealing the first or the secondantigen binding moiety.
 2. The protease-activatable T cell activatingbispecific molecule of claim 1, wherein the masking moiety is: (a)covalently attached to the first antigen binding moiety and reversiblyconceals the first antigen binding moiety; and/or (b) an anti-idiotypicscFv.
 3. The protease-activatable T cell activating bispecific moleculeof claim 2, wherein the masking moiety is covalently attached to theheavy chain variable region of the first antigen binding moiety. 4.(canceled)
 5. The protease-activatable T cell activating bispecificmolecule of claim 1, wherein: (a) the second antigen binding moiety is acrossover Fab molecule wherein either the variable or the constantregions of the Fab light chain and the Fab heavy chain are exchanged;and/or (b) the first antigen binding moiety is a conventional Fabmolecule.
 6. (canceled)
 7. The protease-activatable T cell activatingbispecific molecule of claim 1, comprising not more than one antigenbinding moiety capable of specific binding to CD3.
 8. Theprotease-activatable T cell activating bispecific molecule of claim 1,comprising a third antigen binding moiety, wherein: (a) the thirdantigen binding moiety is a Fab molecule capable of specific binding toa target cell antigen; and/or (b) the third antigen binding moiety isidentical to the second antigen binding moiety.
 9. (canceled)
 10. Theprotease-activatable T cell activating bispecific molecule of claim 1,wherein the second antigen binding moiety is capable of specific bindingto a target cell antigen selected from the group consisting of FolR1,HER1, HER2 and Mesothelin.
 11. The protease-activatable T cellactivating bispecific molecule of claim 1, wherein the first and thesecond antigen binding moiety are fused to each other via a peptidelinker.
 12. The protease-activatable T cell activating bispecificmolecule of claim 1, wherein: (a) the second antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding moiety; (b) the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingmoiety; and/or (c) the T cell activating bispecific moleculeadditionally comprises an Fc domain composed of a first and a secondsubunit capable of stable association. 13-14. (canceled)
 15. Theprotease-activatable T cell activating bispecific molecule of claim 12,wherein the Fc domain is an IgG.
 16. The protease-activatable T cellactivating bispecific molecule of claim 15, wherein the Fc domainexhibits reduced binding affinity to an Fc receptor and/or reducedeffector function, as compared to a native IgG1 Fc domain.
 17. Theprotease-activatable T cell activating bispecific molecule of claim 1,wherein the masking moiety comprises a heavy chain variable regioncomprising: (a) a heavy chain complementarity determining region (CDR H)1 amino acid sequence of SYGVS (SEQ ID NO:26); (b) a CDR H2 amino acidsequence of IIWGDGSTNYHSALIS (SEQ ID NO:27); (c) a CDR H3 amino acidsequence of GITTVVDDYYAMDY (SEQ ID NO:28); and a light chain variableregion comprising: (d) a light chain (CDR L)1 amino acid sequence ofRASENIDSYLA (SEQ ID NO:29); (e) a CDR L2 amino acid sequence of AATFLAD(SEQ ID NO:30); and (f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQID NO:31).
 18. The protease-activatable T cell activating bispecificmolecule of claim 1, wherein the protease cleavable linker comprises atleast one protease recognition sequence.
 19. The protease-activatable Tcell activating bispecific molecule of claim 18, wherein the proteasecleavable linker comprises the protease recognition sequence RQARVVNG(SEQ ID NO:36).
 20. The protease-activatable T cell activatingbispecific molecule of claim 1, wherein: (i) the first antigen bindingmoiety is capable of specific binding to CD3 and comprises a heavy chainvariable region comprising: a) a CDR H1 amino acid sequence of TYAMN(SEQ ID NO:44); b) a CDR H2 amino acid sequence of RIRSKYNNYATYYADSVKG(SEQ ID NO:45); and c) a CDR H3 amino acid sequence of HGNFGNSYVSWFAY(SEQ ID NO:46); and a light chain variable region comprising: d) a CDRL1 amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO:17); e) a CDR L2amino acid sequence of GTNKRAP (SEQ ID NO:18); and f) a CDR L3 aminoacid sequence of ALWYSNLWV (SEQ ID NO:19); and/or (ii) the first antigenbinding moiety is capable of specific binding to CD3 and comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 43 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:
 55. 21. (canceled)
 22. The protease-activatable Tcell activating bispecific molecule of claim 1, wherein: (i) the secondantigen binding moiety is capable of specific binding to FolR1 andcomprises a heavy chain variable region comprising: a) a CDR H1 aminoacid sequence of NAWMS (SEQ ID NO:14); b) a CDR H2 amino acid sequenceof RIKSKTDGGTTDYAAPVKG (SEQ ID NO:15); and c) a CDR H3 amino acidsequence of PWEWSWYDY (SEQ ID NO:16); and a light chain variable regioncomprising: d) a light chain (CDR L)1 CDR L1 amino acid sequence ofGSSTGAVTTSNYAN (SEQ ID NO:17); e) a CDR L2 amino acid sequence ofGTNKRAP (SEQ ID NO:18); and f) a CDR L3 amino acid sequence of ALWYSNLWV(SEQ ID NO:19); or (ii) the second antigen binding moiety is capable ofspecific binding to Mesothelin and comprises a heavy chain variableregion comprising: a) a CDR H1 amino acid sequence of GYTMN (SEQ IDNO:107); b) a CDR H2 amino acid sequence of LITPYNGASSYNQKFRG (SEQ IDNO:108); and c) a CDR H3 amino acid sequence of GGYDGRGFDY (SEQ IDNO:109); and a light chain variable region comprising: d) a CDR L1 aminoacid sequence of SASSSVSYMH (SEQ ID NO:110); e) a CDR L2 amino acidsequence of DTSKLAS (SEQ ID NO:111); and f) a CDR L3 amino acid sequenceof QQWSKHPLT (SEQ ID NO:112).
 23. (canceled)
 24. An idiotype-specificpolypeptide for reversibly concealing an anti-CD3 antigen binding siteof a molecule. 25-32. (canceled)
 33. A pharmaceutical compositioncomprising the protease-activatable T cell activating bispecificmolecule of claim 1 and a pharmaceutically acceptable carrier. 34-37.(canceled)
 38. A method of treating a disease in an individual,comprising administering to said individual a therapeutically effectiveamount of a composition comprising the protease-activatable T cellactivating bispecific molecule of claim
 1. 39. The method of claim 38for treating or delaying progression of cancer, treating or delayingprogression of an immune related disease, or enhancing or stimulating animmune response or function in an individual.